CN111304203B - Promoter Pro-BIUTNT for efficiently driving target gene to be stably expressed in apple protoplast cells - Google Patents

Abstract

The invention discloses a promoter Pro-BIUTNT for efficiently driving a target gene to be stably expressed in an apple protoplast cell, wherein the promoter is a 1307bp nucleotide fragment of a polyubiquitin coding gene starting from A (excluding A) in an ATG (translation initiation site) upwards. Experiments prove that: the Pro-BIUTNT promoter can efficiently and stably drive and regulate 12 representative reference target genes in apple fruit maturation and immune reaction to be expressed in apple protoplast cells, breaks through the technical limitation that partial genes are not expressed and partial genes are weaker in expression when a CaMV35S promoter is applied to the apple protoplast cells, and lays a foundation for applying an apple protoplast cell protein expression technology to apple signal transduction research.

Description

Promoter Pro-BIUTNT for efficiently driving target gene to be stably expressed in apple protoplast cells

Technical Field

The invention relates to the field of plant biotechnology and biochemistry, in particular to a promoter Pro-BIUTNT for efficiently driving a target gene to be stably expressed in apple protoplast cells.

Background

Apple (Malus x domestica Borkh.) is a kind of fruit tree of Rosaceae with great economic value, and is called four fruits in the world together with pear, orange and grape. The molecular mechanism research on the growth and development of apple trees and the formation and regulation of fruit quality enables the tree species to be expected to become a model plant for researching the growth, development, fruit quality formation, disease resistance and immunity of higher perennial woody plants. The method discloses important functional genes and specific signal transduction paths in the apples, and does not leave the efficient and stable expression technology of target genes in apple cells. The target protein can be obtained by applying callus transformation, but the technical method has long period, the research result is easily influenced by the physiological state of cells and somatic variation in the cell culture process, the protein posttranslational modification only in a specific time after protein translation cannot be accurately researched, and the research precision and efficiency are not enough to support the research of apple cell fine signal transduction. The development of a stable, efficient and accurate protein expression system in apple cells is particularly key for revealing important functional genes in apples and revealing molecular mechanisms of apple cell signal transduction on the protein level.

The protoplast cell protein expression technology is a more accurate research system for carrying out cell signal transduction research on the protein level. The efficient and stable expression of target protein in protoplast cell is the precondition of applying the protoplast cell expression technology to the research of gene function and biochemical event in cell signal transduction.

The promoter is a key factor for driving the target gene to express and translate in the cell, and the selection of a proper promoter has a key and determining effect on whether the target gene is expressed or not. However, in the apple protoplast cell, the conventionally used CaMV35S promoter is applied, partial genes cannot be expressed, and partial genes are weakly expressed, so that the characteristic of the CaMV35S promoter hinders the application of the target protein expression technology of the apple protoplast cell to the research of the gene function of the apple.

Disclosure of Invention

The invention aims to provide a promoter for efficiently driving a target gene to be stably expressed in a plant cell and application thereof.

In a first aspect, the invention claims a DNA molecule with promoter function.

The DNA molecule is a) or b) or c) as follows:

a) DNA molecule shown in SEQ ID No. 1;

b) a DNA molecule which has more than 99%, more than 95%, more than 90%, more than 85% or more than 80% of homology with the nucleotide sequence limited by a) and has a promoter function;

c) a DNA molecule which is hybridized with the nucleotide sequence defined by a) or b) under strict conditions and has the function of a promoter.

The stringent conditions may be hybridization in a solution of 6 XSSC, 0.5% SDS at 65 ℃ and washing the membrane once with each of 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS.

In a second aspect, the invention claims recombinant vectors, expression cassettes, recombinant bacteria or transgenic plant cell lines containing the above DNA molecules.

Further, the recombinant vector may be a recombinant expression vector or a recombinant cloning vector.

Further, 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.

Primer pairs for amplifying the whole length of the DNA molecule or any fragment thereof also belong to the protection scope of the invention.

In a third aspect, the invention claims the use of the above DNA molecule as a promoter.

In a fourth aspect, the present invention claims the use of the above DNA molecule or a recombinant vector, expression cassette, recombinant bacterium or transgenic plant cell line containing the above DNA molecule to initiate expression of a target gene.

Further, the promoting target gene expression is promoting target gene expression in a plant cell.

Further, the plant may be a dicot or a monocot.

Still further, the dicot may be an apple plant or a tobacco plant.

The cells may be protoplast cells or leaf epidermal cells.

In a specific embodiment of the invention, the plant cell is an apple protoplast cell.

In another embodiment of the invention, the plant cell is a tobacco lamina epidermal cell.

The application of the DNA molecule or the recombinant vector, the expression cassette or the recombinant bacterium containing the DNA molecule in researching protein interaction also belongs to the protection scope of the invention.

In a fifth aspect, the invention claims a method of expressing a gene of interest.

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

Further, the target gene is expressed in the apple protoplast cells;

the study of protein interaction is to study protein interaction 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.

Further, the apple protoplast cells can be Orin apple callus cells or purplish red 2-4 apple callus cells. 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 on the 10 th day are used for preparing the apple protoplast cells optimally.

In a sixth aspect, the invention claims a method of studying protein interactions.

The method for researching protein interaction claimed by the invention comprises the following steps: the expression of the gene encoding the target protein is promoted by using the DNA molecule as a promoter.

Further, the protein interaction is specifically the protein interaction of AtBAK1 and AtFLS 2.

In any of the above applications or methods, the target gene may be any one or several or all of the following genes: MdERF1, MdERF2, MdERF3, MdERF6, MdEIL2, MdERF98, AtBAK1, AtFLS2, MdBAK1, MdFLS2, MdWRKY29, MdWRKY 33-1.

The invention obtains a nucleotide fragment with promoter function by cloning from arabidopsis thaliana, which is 1307bp nucleotide sequence of polyubiquitin coding gene starting from A (excluding A) in translation initiation site ATG and is named as Pro-BIUTNT. Experiments prove that: the Pro-BIUTNT promoter can efficiently and stably drive and regulate 12 representative reference target genes in apple fruit maturation and immune reaction to be expressed in apple protoplast cells, breaks through the technical limitation that partial genes are not expressed and partial genes are weaker in expression when a CaMV35S promoter is applied to the apple protoplast cells, 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 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. The days in the figure are the days of apple callus cell growth on MS medium used to isolate apple protoplast cells. Scale bar 50 μm.

FIG. 3 is a maternal expression vector construct 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 CaMV35S, 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: carrying out enzyme digestion identification on an expression vector of a reference target gene; d: the dotted line above is the expression vector structure chart of the target gene using CaMV35S, Pro-BIUTNT promoter, the dotted line below is the expression vector structure chart 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 chart of the reference target gene, (right) is the reference target gene set in the expression vector, CCCCTC (. star) is the sequence formed by connecting SmaI and StuI enzyme cutting ends in the expression vector construction, and the sequence exists in the expression vectors of MdBAK1, MdFLS2, MdWRKY29, MdWRKY33-1 and AtFLS2 cloned by SmaI enzyme cutting.

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 parent vector 'plasmid 3' for illustration display; c: cloning nucleotide sequences in 6 apples serving as promoters into an expression vector with 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 CaMV35S 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 CaMV35S promoter and the Pro-BIUTNT promoter are in the propamolComparison of expression effects of the driving target genes MdERF1, MdERF2, MdERF3 and MdERF6 in the protoplast cells. Expression vectors driven by CaMV35S 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 CaMV35S and Pro-BIUTNT promoters in protoplast cells for driving target gene expression for different times; b: comparison of CaMV35S and Pro-BIUTNT promoter driving expression of a gene of interest in apple protoplast cells prepared from callus cells cultured in MS medium for 6 days.

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

FIG. 10 is a diagram showing the structure of an expression vector for the GFP or GFP-tagged target protein MdERF98 driven by CaMV35S, Pro-BIUTNT promoter. a: plasmid 13-vector construction procedure for plasmid 18; b: expression vector structure diagram of GFP (plasmid 13, plasmid 14) driven by CaMV35S, Pro-BIUTNT promoter or GFP-labeled target protein MdERF98 (plasmid 15, plasmid 16); c: binary expression vector structure diagram of the GFP-labeled target protein MdERF98 (plasmid 17, plasmid 18) driven by CaMV35S, Pro-BIUTNT promoter.

FIG. 11 shows the comparison of the expression of the Pro-BIUTNT and CaMV35S promoters driving the target gene GFP or GFP-labeled protein MdERF98 in apple protoplast cells and tobacco leaf epidermal cells. a: Pro-BIUTNT and CaMV35S promoters drive the expression of GFP and MdERF98-GFP in apple protoplast cells to be compared, (I) Pro-BIUTNT and CaMV35S promoters drive the expression of GFP, II) Pro-BIUTNT and CaMV35S promoters drive the expression of MdERF98-GFP, and (III) Pro-BIUTNT and CaMV35S promoters drive the expression of GFP and MdERF98-GFP to be compared in fluorescence signal intensity; b: comparison of expression of MdERF98-GFP in tobacco leaf epidermal cells driven by Pro-BIUTNT and CaMV35S promoters, (I) expression effect of MdERF98-GFP in tobacco leaf epidermal cells driven by Pro-BIUTNT promoters, (II) expression effect of MdERF98-GFP in tobacco leaf epidermal cells driven by CaMV35S promoters, (III) expression of MdERF98-GFP in tobacco leaf epidermal cells driven by Pro-BIUTNT and CaMV35S promoters (left) and number of cells with and without fluorescence signals and number of cells in different fluorescence signal ranges (right).

FIG. 12 is a study of the interaction between two proteins in apple protoplast cells using the Pro-BIUTNT promoter.

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 after Pro before the gene to which the nucleotide fragment belongs, separated by a spacer "-" between them, and the suffix "-2" or "-1" or "-2" after the gene name indicates different nucleotide fragments from the promoter region of the same gene. Pro is English in 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 application, including the promoter, the gene of interest to be driven, 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 literature "Cui F, Wu S, Sun W, Coaker G, Kunkel B, He P, Shan L.2013.the Pseudomonas syringae type III effect AvrRpt2 proteins pathway used by human virus not using the amplification of Arabidopsis itself/induced acid protein turner, Plant Physiology,162: 1018. 1029." the "binary expression vector pCB 302" is described in the second part of materials and methods on page 11 of the literature, publicly available from the applicant of the present patent (university of Shandong agricultural), which is used only for the repetition of experiments related to the present invention and is not used for other purposes.

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 mauve 2-4 apple callus cells (fig. 1 b).

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

1) Origin of Orin apple callus cells

Sources of Orin apple callus cells are described in the references "AN JP, Yao JF, Xu RR, You CX, Wang XF, Hao YJ. apple bZIP transformation factor MdbZIP44 regulated antisense acid-expressed antisense in culture. plant, Cell & Environment,2018, DOI: 10.1111/pce.13393".

2) Cultivation and preservation of Orin apple callus cells

Culturing and storage of the Orin apple callus cells were performed using MS (Murashige-Skooge) medium (M5519, Sigma) supplemented with 1.5mg/L of 2,4-D and 0.4mg/L of 6-BA, subcultured every 2 weeks under the culture conditions: culturing in dark at 24 deg.C.

2. Source, culture and preservation of calli cells of 2-4 purple apples

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 are described in 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 An anti-cellular hormone biosyntheses in wells cultures of red-fleshed applets (Malus sievessi f. nidzwedyana.). Plant Cell Tissue Organ, Culture,2015,120: 325-.

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 are as follows: the culture was incubated at 24 ℃ in 8 hours of light and 16 hours of darkness per day.

Second, separation of apple protoplast cells

The method comprises the steps of taking more than two apple callus cells growing for different days on an MS culture medium added with phytohormone as test materials to separate and prepare apple protoplast cells. 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).

Solution of W5: 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 in 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 that the apple protoplast cells separated from the calli of purple red 2-4 apples, which have been grown on the MS medium supplemented with plant hormones for 5 days, 10 days, 15 days, 20 days and 25 days, respectively, are used as test samples, wherein the apple protoplast cells separated from the calli of purple red apples, which have been grown on the MS medium supplemented with plant hormones for 20 days and 25 days, are microscopically observed, and no evenly scattered protoplast cells are observed, and the separated protoplast cells aggregate into clumps and have fragmented materials (FIG. 2b, 20 days and 25 days), so that no statistics is made on the number of protoplast cells.

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, mauve 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; 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 CaMV35S, Pro-BIUTNT

The construction scheme of the maternal expression vector using both CaMV35S, 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 CaMV35S promoter, and HAs HA (HA: Hemagglutinin) as C-terminal label. 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 CaMV35S 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 the restriction enzymes PstI/SmaI in a system of 15. mu.L by adding 1.5. mu.L of 10 XNEB Cutsmart buffer, 7. mu.L of 100 ng/. mu.L of "plasmid 2", 0.5. mu.L of PstI (NEB, # R3104) and 5.5. mu.L of sterile water, followed by digestion in a metal bath at 37 ℃ for 2 hours, followed by addition of 0.5. mu.L of SmaI (NEB, # R0141S) and finally digestion in a metal bath at 25 ℃ for 2 hours.

2) After completion of the digestion, the digestion product was electrophoresed on 1.5% agarose gel. The electrophoresis results are shown in FIG. 3b (II). The lower arrow indicates the 564bp CaMV35S promoter released from "plasmid 2"; the remaining portion of the expression vector after release of the CaMV35S promoter is indicated by the upper arrow. 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 the 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 T4DNA ligase (NEB, M0202) at 16 ℃ overnight, and the Escherichia coli is transformed.

3) Selecting Escherichia coli for monoclonal culture, extracting plasmid, and cloning Pro-BIUTNT promoter into target clone of 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 "plasmid 3" as a template, two restriction sites 2 and 3 of "plasmid 3" were mutated from SmaI to BamHI and BamHI to SmaI, respectively, using Thermo SCIENTIFIC (GeneArt, a13282) kit and primers listed in table 3, to obtain an expression vector using the Pro-BIUTNT promoter capable of cloning a target gene into the expression vector using the other restriction sites, which was designated "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 "plasmid 3" was mutated from HA to FLAG using Thermo SCIENTIFIC (GeneArt, A13282) kit and the primers listed in Table 3 as a template to obtain an expression vector with the C-terminal tag FLAG, wherein the sequence of the FLAG tag is indicated in italics in the forward primer sequence (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) Cloning was performed using SmaI single restriction, and 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 an amplification product of the AtFLS2 is precipitated by alcohol and dissolved in sterile water, SmaI is used for single enzyme digestion for 2 hours, meanwhile, expression vector with a C-terminal tag of 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 an AtFLS2 fragment digested by SmaI overnight under the action of T4 ligase, and a ligation product is transformed into escherichia coli.

4) The BamHI/StuI enzyme digestion was used to identify the target clone of AtFLS2 open reading frame 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 CaMV35S promoter.

Thirdly, constructing expression vector of test target gene by using CaMV35S, Pro-BIUTNT two types of promoters

The construction scheme of the expression vector of the reference target gene using two types of CaMV35S, 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 CaMV35S promoter and a Pro-BIUTNT promoter; the bold boxes indicate the expression vectors constructed to the Pro-BIUTNT promoter; the thin-line box indicates the expression vector constructed to the CaMV35S 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 sequences of the open reading frames of the 13 target genes are shown in FIG. 4 b. (I) Shown are the amplification results 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 CaMV35S promoter.

2. Construction of expression vector for reference target Gene

Respectively cloning the open reading frame full-length sequences of the 13 reference target genes amplified in the step 1 into an expression vector. The promoter type (CaMV 35S promoter, Pro-BIUTNT promoter), 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 reference gene was cloned, maternal vectors and restriction enzyme 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 square marker test genes (directly facing to the first row above the square marker test genes) into the promoter type of the expression vector, a female parent vector (directly facing to the left first row) and the used enzyme cutting cloning site (directly facing to the left second row); black fill shows genes (Gene 1-Gene 10) constructed into expression vectors using CaMV35S promoter and Pro-BIUTNT promoter, respectively; purple filling shows genes (gene 11 and gene 12) constructed into an expression vector using a Pro-BIUTNT promoter; orange fill shows the gene (gene 13) constructed into the expression vector using the CaMV35S promoter; 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 reference 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 digestion identification (shown by "-) of 6 target genes respectively using CaMV35S promoter and Pro-BIUTNT promoter as driving expression vectors, and restriction enzyme digestion identification (shown by" -) of expression vectors of MdMAPK6 driven by the CaMV35S promoter and AtBAK1 and AtFLS2 expression vectors driven by the Pro-BIUTNT promoter; the gene names are shown in the figure. The number "1" indicates an expression vector using CaMV35S 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 the expression vector which applies the CaMV35S promoter and the Pro-BIUTNT promoter is carried out on the MdERF1, the MdERF2, the MdERF3 and the MdERF 6.

3 clones are selected from expression vectors driven by CaMV35S 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) are shown by enzyme digestion identification results, and all the genes to be tested are cloned into the expression vector. Wherein, the nucleotide sequence of the expression vector of the target gene MdERF1 driven by the Pro-BIUTNT promoter is shown as a sequence 8 in the sequence table.

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 a1, 5-ribulose two phosphate 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-MdBIUTNT and Pro-MdBIUTNT-2 are two nucleotide fragments which are 1539bp and 2501bp respectively from A (excluding A) in the translation initiation site ATG of the MdBIUTNT 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 FIG. 5c (I); Pro-MdRC-1 and Pro-MdRC-2 amplification products are shown by arrows in FIG. 5c (I).

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 MdERF98 amplification.

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 expression vectors respectively to be used as promoters, and respectively 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 subjected to sequencing verification.

The 'plasmid 6' is an intermediate vector in vector construction, and the 'plasmid 7' -the 'plasmid 12' is an expression vector which uses 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 the present 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 of 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.

Secondly, comparing the expression effect of Pro-BIUTNT, Pro-MdBIUTNT-2 promoter and CaMV35S promoter driving target gene in apple protoplast cell

1. Comparison of effects of Pro-BIUTNT and CaMV35S promoters on driving target gene to express 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 CaMV35S 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 MdERF2 is expressed weakly by using CaMV35S 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 CaMV35S 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 CaMV35S promoter, and clear and specific target protein expression signals are obtained by using Pro-BIUTNT promoter (figure 6a, figure 7, figure 9).

MdEIL2 is a nucleotide sequence with longer amino acid coding region than the above 5 ERF transcription factor family genes, CaMV35S is used as promoter, no expression is obtained, Pro-BIUTNT promoter is used, clear and specific target protein expression signal is obtained at the position of target protein (FIG. 6a,

shown).

(3) MdWRKY33-1 and MdWRKY29 are important transcription factors of WRKY family. The expression of MdWRKY33-1 cannot be driven by CaMV35S 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 CaMV35S 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); in addition, 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, and no protein expression signals were obtained using CaMV35S promoter (FIG. 8 b).

Although the CaMV35S 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 CaMV35S 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 CaMV35S 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

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 CaMV35S 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 comparison of Pro-BIUTNT and CaMV35S promoter-driven expression effects of GFP or GFP-labeled target genes in apple protoplast cells and tobacco leaf epidermal cells

Construction of Pro-BIUTNT and CaMV35S promoter-driven GFP or GFP-labeled target gene expression vector

In order to further compare the driving capability of Pro-BIUTNT and CaMV35S promoters, CaMV35S and Pro-BIUTNT promoters used for transfection of apple protoplast cells and tobacco leaf epidermal cells respectively are constructed to drive expression vectors of GFP and GFP-labeled MdERF98, and the structure diagram of the vectors is shown in FIG. 10. The preparation method comprises the following steps:

1. plasmid 15

The GFP fragment was released from the AvrPto-GFP expression vector and recovered by StuI/PstI digestion, and cloned into CaMV 35S: : MdERF98 expression vector (the structure of the expression vector is shown in FIG. 4d), and the CaMV35S promoter-driven MdERF98-GFP expression vector is obtained and named as "plasmid 15" (FIG. 10a, I-step 1).

2. Plasmid 13

Application of Thermo scientfic (GeneArt, a13282) kit and primer F: 5'-GTGAGCAAGGGCGAGGAGCTG-3' and R: 5'-CATGGATCCACGGAGCAAGGG-3', the MdERF98 gene in "plasmid 15" was deleted to obtain an expression vector using CaMV35S promoter to drive GFP, which was designated as "plasmid 13" (FIG. 10a, I-step 2).

3. Plasmid 16

1) The application of primer F: 5' -GAAGGCCTGTGAGCAAGGGC-3' with R: 5' -CGGGATCCCCGGGCGGCCGCTTTACT-3 ' was amplified from the AvrPto-GFP expression vector to obtain GFP fragments with StuI and BamHI cleavage sites at the 5 ' and 3 ' ends, respectively.

2) The cloning was performed by StuI/BamHI digestion, and the GFP fragment was cloned into "plasmid 6" (FIG. 5d for this expression vector), resulting in an expression vector using the Pro-BIUTNT promoter to drive MdERF98-GFP, which was designated as "plasmid 16" (FIG. 10a, II-step 1).

4. Plasmid 14

Application of Thermo scientfic (GeneArt, a13282) kit and primer F: 5'-GTGAGCAAGGGCGAGGAGCTG-3' and R: 5' -CATGGATCCCTGTTAATCAGAAAAACTCAG-3', MdERF98 gene in "plasmid 16" was deleted to obtain Pro-BIUTNT promoter-driven GFP expression vector, which was named "plasmid 14" (FIG. 10a, II-step 2). The underlined bar in the R primer indicates the added BamHI cleavage site, and the italic indicates the ATG translation initiation site.

5. Plasmid 17

1) Applying a primer F: 5' -CGGGATCCATGGAGGGGAAGAGAGGAC-3' with R: 5'-GAAGGCCTCTGTGTTGGTTGCCCCTGCCTAT-3' Pro-BIUTNT constructed from example 2 (III): : the MdERF98 expression vector (the structure of the expression vector is shown in figure 4d) is amplified to obtain the full-length sequence of the MdERF98 open reading frame with BamHI and StuI enzyme cutting sites at the 5 'and 3' ends respectively.

2) The cloning was performed using BamHI, StuI restriction enzymes, MdERF98 was cloned into binary expression vector pCB302(pCB 302: CaMV 35S: : axr2-2FLAG, fig. 10a, III), yielding pCB 302: CaMV 35S: : MdERF98-FLAG (FIG. 10a, III-step 1).

3) The clones were digested with StuI/PstI, and the GFP fragment was cloned from the AvrPto-GFP expression vector into pCB 302: CaMV 35S: : in the MdERF98-FLAG expression vector, the FLAG tag in the MdERF98-FLAG expression vector is replaced by GFP to obtain an expression vector which uses a CaMV35S promoter to drive MdERF98-GFP and is named as 'plasmid 17' (FIG. 10a, III-step 2).

6. Plasmid 18

1) The application of primer F: 5'-GGATCCAAGCTTACTCCAAGAAATATCAAAG-3' and R: 5'-AGCTTGTCACTGGCCGTCGTT-3' the restriction site at the front end of CaMV35S promoter in the binary expression vector pCB302 was mutated from XhoI to BamHI to give the expression vector "plasmid A" (FIG. 10a, IV-step 1).

2) Applying a primer F: 5'-CGGGATCCGATCAGGATATTCTTGTTTAAGATG-3' and R: 5'-AACTGCAGCTGTGTTGGTTGCCCCTGCCTAT-3' the Pro-BIUTNT-MdERF98 whole fragment obtained by amplifying Pro-BIUTNT having BamHI and PstI cleavage sites at 5 'and 3' ends, respectively, from "plasmid 16" and ligating it with MdERF98, and cloning the fragment into "plasmid A" by digestion with BamHI/PstI, to obtain "plasmid B" (FIG. 10a, IV-step 2).

3) The application of primer F: 5' -AACTGCAGGTGAGCAAGGGCGAGGAGC-3' with R: 5' -AACTGCAGCCGGGCGGCCGCTT-3 ' is amplified from an AvrPto-GFP expression vector to obtain a GFP fragment with PstI enzyme cutting sites at the 5 ' and 3 ' ends; the GFP fragment was cloned into "plasmid B" using PstI single-restriction cloning, and the expression vector using the Pro-BIUTNT promoter to drive MdERF98-GFP was obtained and named "plasmid 18" (FIG. 10a, IV-step 3).

Secondly, target protein expression and detection

1. Expression and detection of target protein in apple protoplast cells

According to the method in example 4, the plasmids 13-16 constructed in the first step are transfected into apple protoplast cells, the protoplast cells are extracted after 6 hours of protein expression, and the protoplast cells are placed under an Olympus, BX53 fluorescence microscope to observe the fluorescence signal of the target protein, wherein the wavelength of the excitation wave is 488nm, and the wavelength of the filter absorption wave is 550-590 nm. While observing the MdERF98-GFP fluorescence signal, the nuclei of protoplast cells were stained with 1. mu.g/mL DAPI (C1002, Byunnan) stain, and the fluorescence signal intensities of the nuclear DAPI stains were observed and compared under UV filters.

The results are shown in FIG. 11a, and FIG. 11a (I) shows the comparison of the expression effect of GFP; FIG. 11a (II) is a comparison of the effect of GFP-tagged MdERF98 expression. The results show that: in apple protoplast cells, the expression signals of GFP and GFP-labeled MdERF98 using Pro-BIUTNT promoter were significantly stronger than those using CaMV35S promoter, the fluorescence signal intensity of GFP was 18 times that using CaMV35S promoter, and the fluorescence signal intensity of MdERF98-GFP in nucleus was 11.4 times that using CaMV35S promoter (FIGS. 11a, III). The fluorescent signal intensities of DAPI staining of two cell nucleuses are similar, which indicates that the Pro-BIUTNT promoter is a promoter with stronger driving capability than CaMV 35S.

2. Expression and detection of target protein in epidermal cells of tobacco leaves

The plasmid 17 and the plasmid 18 constructed in the first step are transfected into tobacco leaf epidermal cells respectively. The method comprises the following specific steps: the plasmid 17 and the plasmid 18 are respectively transformed into agrobacterium tumefaciens competent LBA4404, a single clone is picked up, LB liquid culture medium (kanamycin 50mg/L and rifampicin 50mg/L) containing antibiotics is used for culturing for 24 hours, 200 mu L of bacterial liquid is absorbed and added into 10mL of LB liquid culture medium containing 200 mu M Acetosyringone (AS) and 10mM MES for further culturing for 12 hours. After the culture, the cells were centrifuged at 5000rpm for 10min, and the cells were collected and suspended in a suspension (10mM MgCl. was used)₂10mM MES, 200. mu.M AS) and the OD₆₀₀The resulting mixture was adjusted to 0.6 and allowed to stand at room temperature for 3 hours. Should be takenThe bacterial liquid is injected into the epidermal cells of the leaves of the native tobacco which grow for 25 days by using a sterile injector, the injected leaves are marked by using a marking pen, and then the plants are transferred to the condition of 28 ℃ for culture. After culturing for 2-3 days, gently tearing off the lower epidermis of the tobacco leaf by using forceps, flatly paving on a glass slide, lightly covering a cover glass, and observing the fluorescence signal of the target protein by microscopic examination under an Olympus, BX53 fluorescence microscope. The wavelength of the excitation light is 488nm, and the filter absorbs wavelength 550-590 nm.

The results are shown in FIG. 11b, (I) in FIG. 11b shows the effect of using Pro-BIUTNT promoter to drive the expression of MdERF98-GFP in tobacco leaf epidermal cells, and (II) in FIG. 11b shows the effect of using CaMV35S promoter to drive the expression of MdERF98-GFP in tobacco leaf epidermal cells. The results show that: in the epidermal cells of the tobacco leaves, the number of cells with fluorescence signals in the cell nucleus when the Pro-BIUTNT promoter is applied is obviously more than that when the CaMV35S promoter is applied, and the intensity of the fluorescence signals in the cell nucleus when the Pro-BIUTNT promoter is applied is obviously stronger than that when the CaMV35S promoter is applied.

In total 170 cells, 35 of which had a fluorescent signal, were observed in the field using the Pro-BIUTNT promoter; in contrast, there were 167 cells in total in the field observed using the CaMV35S promoter, 1 of which had a fluorescent signal (FIG. 11b, III-left) and an intensity of the fluorescent signal was weaker than that of most of the cells using the Pro-BIUTNT promoter (FIG. 11b, III-right).

The above results illustrate that: the Pro-BIUTNT promoter is a promoter with greater driving ability than the CaMV35S promoter.

Example 6 investigation of the interaction between two proteins in apple protoplast cells Using the Pro-BIUTNT promoter

Two proteins on plant cell membranes AtBAK1(NM _119497.5) and AtFLS2(NM _001344672.1) are able to interact under stimulation by a flg22 short peptide consisting of 22 conserved amino acids of bacterial flagellin (see references: Chinchilla D, Zipfel C, Robarzek S, Kemmerling B, N ü rnberger T, Jones JDG, Felix G, Boller T.A flagellin-induced complex of the receptor FLS2 and BAK1 initiates plant destination Nature,2007,448:497-500.), which is an important event in plant immune signal transduction.

The Pro-BIUTNT driven AtBAK1 constructed in example 2 was co-transfected into and expressed in apple protoplast cells with AtFLS2 expression vector (see FIG. 4d below the dotted line and FIG. 4a (II)), and then apple protoplast cells were treated with flg22 to investigate the interaction between AtBAK1 and AtFLS2 by co-immunoprecipitation. Specific procedures for co-immunoprecipitation are described in references "Lu D, Wu S, Gao X, Zhang Y, Shan L, He P.A. receptor-like cytoplasmatic kinase, BIK1, associates with a flexible receptor complex to initial plant immunity. Proc Natl Acad Sci USA,2010,107: 496-501".

The results are shown in FIG. 12. The results show that: stimulation with flg22 induced an interaction between AtBAK1 and AtFLS 2. The application of the Pro-BIUTNT promoter is demonstrated to enable the study of protein interactions in apple protoplast cells.

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> promoter Pro-BIUTNT for efficiently driving target gene to be stably expressed in apple protoplast cells

<160>8

<170>PatentIn version 3.5

<210>1

<211>1307

<212>DNA

<213>Artificial Sequence

<400>1

gatcaggata ttcttgttta agatgttgaa ctctatggag gtttgtatga actgatgatc 60

taggaccgga taagttccct tcttcatagc gaacttattc aaagaatgtt ttgtgtatca 120

ttcttgttac attgttatta atgaaaaaat attattggtc attggactga acacgagtgt 180

taaatatgga ccaggcccca aataagatcc attgatatat gaattaaata acaagaataa 240

atcgagtcac caaaccactt gcctttttta acgagacttg ttcaccaact tgatacaaaa 300

gtcattatcc tatgcaaatc aataatcata caaaaatatc caataacact aaaaaattaa 360

aagaaatgga taatttcaca atatgttata cgataaagaa gttacttttc caagaaattc 420

actgatttta taagcccact tgcattagat aaatggcaaa aaaaaacaaa aaggaaaaga 480

aataaagcac gaagaattct agaaaatacg aaatacgctt caatgcagtg ggacccacgg 540

ttcaattatt gccaattttc agctccaccg tatatttaaa aaataaaacg ataatgctaa 600

aaaaatataa atcgtaacga tcgttaaatc tcaacggctg gatcttatga cgaccgttag 660

aaattgtggt tgtcgacgag tcagtaataa acggcgtcaa agtggttgca gccggcacac 720

acgagtcgtg tttatcaact caaagcacaa atacttttcc tcaacctaaa aataaggcaa 780

ttagccaaaa acaactttgc gtgtaaacaa cgctcaatac acgtgtcatt ttattattag 840

ctattgcttc accgccttag ctttctcgtg acctagtcgt cctcgtcttt tcttcttctt 900

cttctataaa acaataccca aagagctctt cttcttcaca attcagattt caatttctca 960

aaatcttaaa aactttctct caattctctc taccgtgatc aaggtaaatt tctgtgttcc 1020

ttattctctc aaaatcttcg attttgtttt cgttcgatcc caatttcgta tatgttcttt 1080

ggtttagatt ctgttaatct tagatcgaag acgattttct gggtttgatc gttagatatc 1140

atcttaattc tcgattaggg tttcataaat atcatccgat ttgttcaaat aatttgagtt 1200

ttgtcgaata attactcttc gatttgtgat ttctatctag atctggtgtt agtttctagt 1260

ttgtgcgatc gaatttgtcg attaatctga gtttttctga ttaacag 1307

<210>2

<211>1539

<212>DNA

<213>Artificial Sequence

<400>2

ccttgaaaac aaattatttt ttcggttttt aattttttgg ggaggggagg cagattctct 60

gcccttctac ttttcgtgcc ctttcatgct cttttgtttg tgtgataatc gttaaatcac 120

gttaacattt tatattacta atctttttta tcttattatc tttataaaaa aattataaaa 180

tattaatgtg atttaaccgt aactacacaa aataagaggg tacggaaggt aggagggcag 240

acaatctacc tcctcacgcg cacccttttg accattcaaa aggcccctgc acatttctgt 300

tcattgattt ctcttttttg ggttttttca acgaatgata ttatttcctt aagaggataa 360

atagtgttag cctcacaatt gactagtaat aatgttgttc aaatttctcc ttggcgaaaa 420

tcgaacttaa gatttctttc ttgccaataa agagaaatac cactaaatca tagtagtagc 480

tcacctcctt ctacttgtat acaacttttt aaattacttt tcaagttagt ttcaagtact 540

tatttggtat tactatttat aaaaactaaa gtcaaaatta acatatgaat ttgggaatga 600

ggatggcacg cccgaacttg atactcttag ttgccatccc tactctctcc tctttctccc 660

tcactctcct tgatattctt ctcccgtctc ttctccgata ttctttcgcc attttctctc 720

tcttctcttt gaatttccaa gttcggataa tgttacccat tacactgtag gatgagatgt 780

ttttactttt ctaattgttg tattttgaaa acaattttaa atcacataca taaacaagtt 840

ttttattctt ttaagaagta ttcctcaaaa atgttttgag gaaagatgta cctttttata 900

taagatacca aacagcttct tatttttgtc atataatttc acattaagat gtactattca 960

cacatctttt tatttctcac atattccttg ttaattttta tccgtcgatc ttcttcaatt 1020

tatcaaaccc gatacatgaa aattaaaaag gtgtgtgaaa ggcaaaaaag tgtgtgaata 1080

tcagatctct ttagtaatcc attagcaggt catatacgat attgacgcgt gtaatccaca 1140

taacctgtca acgtagcatt ctgcaagtcc aaacaaatta ggaatccaat taccttcaca 1200

agcaaggaaa ggggcaacaa aaatccaaga tagagcaggt caccttctcc ctcatcaaat 1260

aaagaaaact aaaaatcaga ggtcaacaat tgttggcact tgcccaccta gaaacatctg 1320

ggccgttcgt ttcactgact tttctggtcc acgcgttcct cgtccactcc ggaatccaac 1380

ctaaaaatgg gatttgagtc ggctcatcct cccctatata aactcctcac ccccctccac 1440

tcttcctcac aattctctca acaattctcg cccatcgtcc ccaaatctct ctcaaatccc 1500

taatttcttt cgaattacaa atccccaatt tctgaaacc 1539

<210>3

<211>2501

<212>DNA

<213>Artificial Sequence

<400>3

ggatttatgg ctgtggatcg cccagctttt cctgggtcga ggaatatgct tgatcatcat 60

agagacatga ggctggacat agacaacatg agttatgagg taacgtcaat attcctgtct 120

attatatttc ttgaactgtc tgaacaaaaa ggaactccta gcactaggcg aaagaatagg 180

aaatgccttt atttttctct tcctctgact ttcatgtatt ctgaatttct cgaaatgtcc 240

gaacaaatag gaactcctcg cactaagcga aaggattggg aatgtcagca caggcttgtc 300

tgaagatttg ataccgaagt gtttaacaga aacaatatat tgttcatcag atcaaatcca 360

ggatgaaggt tcatgtgcaa tttgcctggt aagagtcttc cacttgaaac cggaaattat 420

aagattgccg tttccattag tgtggtttta attttatctt agcgcttttt agaccaattg 480

gactgtaaat tgcttaactg ctggaaatct aacttgtata taacccgatt gacaggaaga 540

gtacaatgac agggatgatg ttggtgcatt gaaaagttgc ggtcacgatt accatgtgag 600

ctgcatcaag aagtggttgt caatgaaaaa ttcttgtcca atctgcaaag gttgtgctct 660

tcctgataat atgaaggaga aataatttaa tcacgtcgtt agcgcttcat tatattacca 720

ttcttgtata tatatttaga gataatcccg aaaacggaag aaacaatact tgtttcgtat 780

tatgtaaagg gagtgttagc caatttaaat taatttgttt caggcttcca tttaaatgtt 840

gtgaagcctt tttgcaattg ttgttgttcc gtatatggct ctagtatata gttttttttt 900

cttctaattt tatgattcct tttcaaaact gagaagctag tttatgatgg aaaggggaaa 960

accttgaaaa caaattattt tttcggtttt taattttttg gggaggggag gcagattctc 1020

tgcccttcta cttttcgtgc cctttcatgc tcttttgttt gtgtgataat cgttaaatca 1080

cgttaacatt ttatattact aatctttttt atcttattat ctttataaaa aaattataaa 1140

atattaatgt gatttaaccg taactacaca aaataagagg gtacggaagg taggagggca 1200

gacaatctac ctcctcacgc gcaccctttt gaccattcaa aaggcccctg cacatttctg 1260

ttcattgatt tctctttttt gggttttttc aacgaatgat attatttaca ttaagaggat 1320

aaatagtgtt tagcctcaca attgactagt aataatgttg ttcaaatttc tccttggcga 1380

aaatcgaact taagatttct ttcttgccaa taaagagaaa taccactaaa tcatagtagt 1440

agctcacctc cttctacttg tatacaactt tttaaattac ttttcaagtt agtttcaagt 1500

acttatttgg tattactatt tataaaaact aaagtcaaaa ttaacatatg aatttgggaa 1560

tgaggatggc acgcccgaac ttgatactct tagttgccat ccctactctc tcctctttct 1620

ccctcactct ccttgatatt cttctcccgt ctcttctccg atattctttc gccattttct 1680

ctctcttctc tttgaatttc caagttcgga taatgttacc cattacactg taggatgaga 1740

tgtttttact tttctaattg ttgtattttg aaaacaattt taaatcacat acataaacaa 1800

gttttttatt cttttaagaa gtattcctca aaaatgtttt gaggaaagat gtaccttttt 1860

atataagata ccaaacagct tcttattttt gtcatataat ttcacattaa gatgtactat 1920

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

<210>8

<211>6234

<212>DNA

<213>Artificial Sequence

<400>8

taacaaaata ttaacgttta caattttatg gtgcactctc agtacaatct gctctgatgc 60

cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg 120

tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca 180

gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt 240

tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg 300

aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct 360

catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat 420

tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc 480

tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg 540

ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg 600

ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga 660

cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta 720

ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc 780

tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc 840

gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg 900

ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc 960

aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca 1020

acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct 1080

tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat 1140

cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg 1200

gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat 1260

taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact 1320

tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat 1380

cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc 1440

ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct 1500

accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg 1560

ctcagcagag cgcagatacc aaatactgtc tctagtgtag cgtagttagg ccaccacttc 1620

aagaactctg tagcacgcct acatacctcg ctctgctaat cctgttacca gtggctgctg 1680

ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg 1740

cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct 1800

acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga 1860

gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc 1920

ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg 1980

agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg 2040

cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt 2100

tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc 2160

gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac 2220

gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc 2280

ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg 2340

caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat 2400

aacaatttca cacaggaaac agctatgacc atgattacgc caagcttatc gtcgacctgc 2460

aggatcagga tattcttgtt taagatgttg aactctatgg aggtttgtat gaactgatga 2520

tctaggaccg gataagttcc cttcttcata gcgaacttat tcaaagaatg ttttgtgtat 2580

cattcttgtt acattgttat taatgaaaaa atattattgg tcattggact gaacacgagt 2640

gttaaatatg gaccaggccc caaataagat ccattgatat atgaattaaa taacaagaat 2700

aaatcgagtc accaaaccac ttgccttttt taacgagact tgttcaccaa cttgatacaa 2760

aagtcattat cctatgcaaa tcaataatca tacaaaaata tccaataaca ctaaaaaatt 2820

aaaagaaatg gataatttca caatatgtta tacgataaag aagttacttt tccaagaaat 2880

tcactgattt tataagccca cttgcattag ataaatggca aaaaaaaaca aaaaggaaaa 2940

gaaataaagc acgaagaatt ctagaaaata cgaaatacgc ttcaatgcag tgggacccac 3000

ggttcaatta ttgccaattt tcagctccac cgtatattta aaaaataaaa cgataatgct 3060

aaaaaaatat aaatcgtaac gatcgttaaa tctcaacggc tggatcttat gacgaccgtt 3120

agaaattgtg gttgtcgacg agtcagtaat aaacggcgtc aaagtggttg cagccggcac 3180

acacgagtcg tgtttatcaa ctcaaagcac aaatactttt cctcaaccta aaaataaggc 3240

aattagccaa aaacaacttt gcgtgtaaac aacgctcaat acacgtgtca ttttattatt 3300

agctattgct tcaccgcctt agctttctcg tgacctagtc gtcctcgtct tttcttcttc 3360

ttcttctata aaacaatacc caaagagctc ttcttcttca caattcagat ttcaatttct 3420

caaaatctta aaaactttct ctcaattctc tctaccgtga tcaaggtaaa tttctgtgtt 3480

ccttattctc tcaaaatctt cgattttgtt ttcgttcgat cccaatttcg tatatgttct 3540

ttggtttaga ttctgttaat cttagatcga agacgatttt ctgggtttga tcgttagata 3600

tcatcttaat tctcgattag ggtttcataa atatcatccg atttgttcaa ataatttgag 3660

ttttgtcgaa taattactct tcgatttgtg atttctatct agatctggtg ttagtttcta 3720

gtttgtgcga tcgaatttgt cgattaatct gagtttttct gattaacagg gatccatgtg 3780

tggtggtgct atcatttccg acttcatcgc cgtcaagcgc gccctgaagc tgacggcgga 3840

ggacctctgg tcagatcttg acaccatctc tgacctcctt ggcatagact actccaacag 3900

catcaacaaa cagccggaga atcacaaggt ggtccaaaag ccgaaaccat ctatcaccaa 3960

agtgacaagt gatgagaagc ccaagcaggc gagcgggtct gctgctgccg caaaaggcaa 4020

gagagtgagg aaaaacgtgt acagaggaat aaggcagagg ccgtggggca aatgggcggc 4080

tgagattcgc gacccctaca aaggcgtccg ggtctggctc ggcacctatg acaccgctga 4140

ggaagccgcc cgcgcttacg atgaagccgc cgtgcgcatc cgcggggaca aggccaagct 4200

caactttgcc caaccaccat cctcttctcc gcttccatct ctggcgccgg atacgccgcc 4260

gccgacaaag aggcggtgca ttgttgctga gtcaactcgg gtggagccga ctcaaccgag 4320

tttccagacc ggttcttact attatgatcc attatatcac ggcggtggtg gtggggaaat 4380

gtatgctaag aaagaggtgg cgggtgggga cgaggtggtg gtagagcagc tgagtcaggt 4440

ggtgagtgga agcggagagt cggactcgtt gtacctgtgg atgctggatg acctggtggc 4500

atatcagcaa caagggcagc ttctgtatag gccttaccca tacgacgttc cagactacgc 4560

tggttaccca tacgacgttc cagactacgc ttgacccggg gaatttcccc gatcgttcaa 4620

acatttggca ataaagtttc ttaagattga atcctgttgc cggtcttgcg atgattatca 4680

tataatttct gttgaattac gttaagcatg taataattaa catgtaatgc atgacgttat 4740

ttatgagatg ggtttttatg attagagtcc cgcaattata catttaatac gcgatagaaa 4800

acaaaatata gcgcgcaaac taggataaat tatcgcgcgc ggtgtcatct atgttactag 4860

atcgggaatt cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc 4920

caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc 4980

cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggcg cctgatgcgg 5040

tattttctcc ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag caaccatagt 5100

acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg 5160

ctacacttgc cagcgcctta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca 5220

cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta 5280

gtgctttacg gcacctcgac cccaaaaaac ttgatttggg tgatggttca cgtagtgggc 5340

catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg 5400

gactcttgtt ccaaactgga acaacactca actctatctc gggctattct tttgatttat 5460

aagggatttt gccgatttcg gtctattggt taaaaaatga gctgatttaa caaaaattta 5520

acgcgaattt taacaaaata ttaacgttta caattttatg gtgcactctc agtacaatct 5580

gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct 5640

gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct 5700

gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga 5760

tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca 5820

cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata 5880

tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga 5940

gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc 6000

ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg 6060

cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc 6120

ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat 6180

cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct caga 6234

Claims (5)

1, DNA molecules or recombinant vectors, expression cassettes, recombinant bacteria or transgenic plant cell lines containing the DNA molecules are applied to starting target gene expression;

the target gene expression is initiated in plant cells;

the plant is an Malus plant or a Nicotiana plant;

the cells are protoplast cells or leaf epidermal cells;

the DNA molecule is shown as SEQ ID No. 1.

2. Use according to claim 1, characterized in that: the target gene is any one or several or all of the following genes: MdERF1, MdERF2, MdERF3, MdERF6, MdEIL2, MdERF98, AtBAK1, AtFLS2, MdBAK1, MdFLS2, MdWRKY29, MdWRKY 33-1.

3. A method of expressing a gene of interest comprising the steps of: using the DNA molecule shown in SEQ ID No.1 as a promoter to start the expression of a target gene; the expression target gene is expressed in the apple protoplast cell.

4. The method of claim 3, wherein: 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.

5. The method of claim 3, wherein: the target gene is any one or several or all of the following genes: MdERF1, MdERF2, MdERF3, MdERF6, MdEIL2, MdERF98, AtBAK1, AtFLS2, MdBAK1, MdFLS2, MdWRKY29, MdWRKY 33-1.

Images