CN113403314B - Corn drought inducible promoter ZmOMAp1730 and application thereof - Google Patents

Corn drought inducible promoter ZmOMAp1730 and application thereof Download PDF

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CN113403314B
CN113403314B CN202110709483.2A CN202110709483A CN113403314B CN 113403314 B CN113403314 B CN 113403314B CN 202110709483 A CN202110709483 A CN 202110709483A CN 113403314 B CN113403314 B CN 113403314B
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于好强
丁磊
庞浩婉
刘媛
付凤玲
李晚忱
杨青青
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Sichuan Agricultural University
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Abstract

The invention discloses a corn drought inducible promoter ZmOMAp1730 and application thereof, belonging to the technical field of agricultural biology. Specifically discloses an obtained corn drought inducible promoter ZmOMAp1730, the nucleotide sequence of which is shown as SEQ ID NO: 1, 1-1730 th base sequence. Also discloses a primer for amplifying the corn inducible promoter ZmOMAp1730 and application of the corn inducible promoter ZmOMAp 1730. Experiments prove that the ZmOMAp1730 gene promoter responds to drought stress and is positively regulated by the drought stress, and the discovery has important significance for plant drought regulation and drought-resistant breeding.

Description

Corn drought inducible promoter ZmOMAp1730 and application thereof
Technical Field
The invention relates to the technical field of agricultural biology, in particular to a corn drought inducible promoter ZmOMAp1730 and application thereof.
Background
Promoters of plant genes are classified into constitutive promoters, inducible promoters and tissue-specific promoters according to the expression modes of the genes. Among them, constitutive promoters are widely used in agricultural biotechnology, such as CAM35S promoter and maize Ubiquitin-1 promoter, and although these promoters are already used, constitutive promoters can promote the continuous and stable high expression of target genes in all tissues of plants, causing the excessive consumption of substances and energy in cells, and the temporal and spatial properties of target gene expression cannot be effectively controlled, and causing the original metabolic balance disorder in plants, so that the normal growth of plants is hindered, and thus there are certain defects in practical application. Repeated driving of multiple foreign gene expression by the same constitutive promoter may also cause gene silencing or co-suppression phenomena. Therefore, it is necessary to induce expression of promoters specifically for different tissues and organs or under specific conditions, so that different promoters can be selected to induce expression of a desired gene according to needs.
Although there are reports on inducible promoters of maize, the functions of different genes may be different, and the functions of all genes are not known by human, and drought is an important factor affecting the growth and yield of maize, so that the provision of an inducible promoter capable of regulating drought stress of maize is very important for solving the problems of maize growth and yield.
Disclosure of Invention
The invention aims to provide a corn drought inducible promoter ZmOMAp1730 and application thereof, so as to solve the problems in the prior art, and experiments prove that the ZmOMAp1730 gene promoter responds to drought stress and is positively regulated by the drought stress, so that the discovery has important significance for plant drought regulation and drought-resistant breeding.
In order to achieve the purpose, the invention provides the following scheme:
the invention provides an obtained corn drought inducible promoter ZmOMAp1730, the nucleotide sequence of which is shown as SEQ ID NO: 1, 1-1730 th base sequence.
The invention also provides a primer for amplifying the corn drought inducible promoter ZmOMAp1730, and the nucleotide sequence of the primer is shown as SEQ ID NO: 2 and the upstream primer shown in SEQ ID NO: 3 as shown in the figure.
Preferably, the amplification procedure is a pre-denaturation at 94 ℃ for 3 min; denaturation at 98 ℃ for 10s, annealing at 68 ℃ for 10s, extension at 72 ℃ for 2min, and 30 cycles; further extension at 72 deg.C for 5 min; keeping at 4 ℃.
Preferably, the amplification system comprises: 5.0. mu.L of 5 XPrimeSTAR Buffer, 2.0. mu.L of dNTP mix, 0.5. mu.L of each of the upstream primer and the downstream primer, 0.2. mu.L of plasmid, 0.25. mu.L of high fidelity enzyme, ddH2O was supplemented to 25.0. mu.L.
The invention also provides an application of the corn drought inducible promoter ZmOMAp1730 or the expression vector or the primer, which is applied to the application shown in the following (a) or (b):
(a) the application in regulating and controlling plant drought stress;
(b) application in plant drought-resistant breeding.
Preferably, the plants include maize, tobacco and arabidopsis.
The invention discloses the following technical effects:
according to the invention, through a plant promoter cis-acting element analysis website, according to the cis-acting element position related to drought in the promoter, a primer is designed near the cis-acting element to amplify 5' -end deletion sequences of ZmOMA gene promoters with different lengths, and the result shows that the GUS activity of a 1730bp promoter-initiated GUS expression vector is highest; and then, the expression vector of the sequence is subjected to functional verification in tobacco and arabidopsis thaliana, and the fact that the ZmOMAp1730 gene promoter responds to drought stress and is positively regulated and controlled by the drought stress is verified. Therefore, the method can provide theoretical basis and data support for plant drought control and drought-resistant breeding.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 is an expression pattern analysis of ZmOMA1 gene; and represents p <0.05, p <0.01, respectively;
FIG. 2 is a sequence analysis and primer design of ZmOMA gene promoter; the core elements TATA-box and CAAT-box are shown in dark and light gray backgrounds, respectively;
FIG. 3 is an electrophoresis diagram showing the amplification of different fragments of the ZmOMA1 gene promoter;
FIG. 4 shows the initiation of GUS analysis by different promoter fragments; GUS histochemical staining; GUS quantitative analysis; and represents p <0.05, p <0.01, respectively;
FIG. 5 is a ZmOMAp1730 promoter activity transient expression analysis under drought treatment; GUS histochemical staining; GUS quantitative analysis; and represents p <0.05, p <0.01, respectively;
FIG. 6 is a ZmOMAp1730 start-up activity stable expression analysis under drought treatment; GUS histochemical staining; and B, GUS quantitative analysis. And represents p <0.05, p <0.01, respectively.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.
Example 1 acquisition and application of maize drought-inducible promoter ZmOMAp1730
1. ZmOMA Gene expression Pattern analysis
When the corn C01 inbred line seeds are cultured to a two-leaf one-heart period, selecting plants with regular and consistent growth, carrying out 20% PEG drought stress treatment, respectively sampling leaves and roots at the treatment time points of 0, 3, 6, 12 and 24h, repeatedly sampling for 3 times in each treatment, immediately carrying out liquid nitrogen quick-freezing grinding after sampling, and storing at-80 ℃. RNA extraction Using Trizol (TaKaRa) kit, followed by PrimeScriptTMThe RT reagent Kit with gDNA Eraser Kit (TaKaRa) reverse transcribes into cDNA.
Primers specific to the gene of interest were designed and GAPDH was used as the reference gene (table 1). qRT-PCR was performed with SYBGreen fluorescent dye, the reaction system in Table 2 was loaded, CFX96TMAmplification on a type quantitative PCR instrument (Bio-Rad, USA). The amplification cycle program is 30s at 95 ℃; 5s at 95 ℃; 30s at 55-65 ℃; the cycle was 39 times, and after the last temperature cycle, the temperature was increased from 65 ℃ to 95 ℃ at a rate of 0.5 ℃ per second for dissolution curve analysis. All amplifications are repeated for 3 times, and after the reaction is finished, the sequence is 2-ΔΔCTThe method calculates test data and analyzes the relative expression quantity of the target gene.
TABLE 1 qRT-PCR primer information
Figure BDA0003132904860000051
TABLE 2 qRT-PCR reaction System
Figure BDA0003132904860000052
Figure BDA0003132904860000061
2. ZmOMA Gene promoter cis-element analysis
The nucleic acid sequence 2000bp before the transcription start site of the ZmOMA gene was downloaded as its promoter sequence. Plant promoter cis-acting element analysis the site plantarte (http:// bioinformatics. psb. element. be/webtools/plantarte/html /) was used to analyze the cis-acting element of the promoter. According to the position of a cis-acting element related to drought in the promoter, a primer is designed near the cis-acting element to amplify the deletion sequence of the 5' end of the ZmOMA gene promoter with different lengths.
3. Promoter cloning and vector construction
Taking corn seedling leaves in the five-leaf stage, carrying out liquid nitrogen quick-freezing grinding, and extracting corn genomic DNA by using a CTAB method. The amplification primer of ZmOMA gene promoter is designed by using NCBI website Primeblast, according to the multiple cloning site of dicotyledonous plant high-efficiency expression vector pRI201-AN-GUS (SEQ ID NO: 9), the Hind III (AAGCTT) cleavage site is introduced into the upstream primer, the Xba I (TCTAGA) site is introduced into the downstream primer, and the specific sequence is shown in Table 3:
TABLE 3 construction of dicotyledonous expression vector primer design
Figure BDA0003132904860000062
Figure BDA0003132904860000071
Samples were added to the reaction system shown in Table 4 and amplified. The temperature cycling program was: 3min at 94 ℃; annealing at 98 deg.C for 10s, 68 deg.C for 10s, and 72 deg.C for 2min for 30 cycles; 5min at 72 ℃; keeping at 4 ℃.
TABLE 4 PCR amplification reaction System
Figure BDA0003132904860000072
And recovering the PCR product. The recovered product and pRI201-AN-GUS plasmid were subjected to double digestion, and the samples were loaded according to the reaction system in Table 5. And (3) uniformly mixing the enzyme digestion system, placing the mixture in a water bath kettle at the temperature of 30 ℃, carrying out enzyme digestion for 6-8 h, adding the enzyme digestion product into a loading buffer solution, carrying out direct electrophoresis, recovering the enzyme digestion product, and carrying out enzyme digestion fragment connection according to the table 6.
TABLE 5 Dual enzyme digestion sample addition System
Figure BDA0003132904860000073
TABLE 6 ligation reaction System
Figure BDA0003132904860000074
The ligation product was transformed into E.coli and tested by colony PCR. After the extracted recombinant plasmid is identified by restriction endonuclease double digestion, the recombinant plasmid bacterial liquid is sent to a sequencing company for DNA sequence sequencing identification, and the plasmid with correct sequencing is converted into tobacco in the next step.
4. Transient expression in tobacco
In order to research the activity difference and key elements among ZmOMA gene promoters with different lengths, the constructed vector is transformed into tobacco by an agrobacterium-mediated method for transient expression.
4.1 competent preparation of Agrobacterium
(1) The preserved Agrobacterium EHA105 was streaked on YEP plates containing 50mg/L rifampicin (Rif), and cultured at 28 ℃ for 36-48 hours.
(2) And selecting a single colony, inoculating the single colony in 1mL of YEP liquid culture medium containing Rif, and carrying out shake culture at the temperature of 28 ℃ for 12-16 h at 200 r/min.
(3) Transferring 1mL of the bacterial solution into 100mL of YEP liquid culture medium containing Rif, performing shake culture at 200r/min and 28 ℃ to OD600=0.4~0.6。
(4) Transferring into a 50mL precooled sterile centrifuge tube, centrifuging for 5min at 4 ℃ and 5000r/min, and collecting thalli.
(5) 10mL of pre-cooled 0.15mol/L NaCl solution was added to suspend the cells,standing on ice for 10 min; 2mL of 0.02mol/L CaCl was added2And (3) solution.
(6) Centrifuging at 4 deg.C and 5000r/min for 5min, and collecting thallus; 1mL of precooled 0.02mol/L CaCl was added2The solution was suspended in the bacteria and left on ice for 10 min.
(7) Subpackaging into sterile tubes at 200 μ L/tube, quickly freezing in liquid nitrogen for 1min, and freezing at-70 deg.C.
4.2 transformation of Agrobacterium with expression vector the following steps were performed:
(1) 200. mu.L of Agrobacterium-infected cells were thawed on ice.
(2) Adding 1 μ g of the constructed carrier, flicking, mixing, and ice-cooling for 30 min.
(3) Quick freezing with liquid nitrogen for 1min, water bathing at 37 deg.C for 5min, adding 700 μ L YEP liquid culture medium containing Rif (50mg/L), and slow shaking culturing at 28 deg.C for 4 h.
(4) The culture was spread on YEP plates containing Kana and Rif and incubated at 28 ℃ for about 36-48 h. Selecting single clone to be cultured in YEP liquid culture medium containing Kana (50mg/L) and Rif (50mg/L) at 28 ℃ overnight, and carrying out PCR detection on bacteria liquid.
4.3 Agrobacterium transformation of tobacco
(1) Agrobacterium containing the recombinant plasmid was streaked on YEP plates containing kanamycin (Kana) and rifampicin (Rif), and cultured at 28 ℃ for 2-3 days.
(2) A single colony of Agrobacterium was picked and inoculated in 3mL of YEP liquid medium containing Kana and Rif, cultured at 28 ℃ at 200r/min, and shaken overnight.
(3) Inoculating 1mL of overnight-cultured starting Agrobacterium strain in 100mL of liquid YEP medium (containing Kana and Rif), and shake-culturing at 28 deg.C to OD600The value is 1.2 to 1.5.
(4) Centrifuging at 4 deg.C for 10min at 5000r/min to collect cells, suspending thallus with tobacco injection (Table 7) and adjusting OD600The value is 0.8 to 1.0.
(5) After standing and activating for 3h at 28 ℃, injecting the heavy suspension into the back of the tobacco leaves by using a sterile syringe, and marking injection positions.
(6) The tobacco injected with the agrobacterium is cultured for 48 hours under the conditions of 25 ℃ and 14/10 hours of photoperiod, and then used for GUS histochemical staining and GUS activity quantitative analysis.
TABLE 7 tobacco injection formulation (200mL)
Figure BDA0003132904860000091
Figure BDA0003132904860000101
5. GUS histochemical staining and Activity determination
5.1 GUS histochemical staining
And (3) dyeing the treated transgenic tobacco leaves with a proper amount of GUS histochemical staining reaction solution overnight. And sucking out the staining solution the next day, and adding 70% ethanol for decoloring for 3-5 times until the background is white as a negative control. Visually observed and photographed.
5.2 GUS Activity assay
The GUS enzyme activity determination method mainly refers to a Jefferson method. The method takes 4-methylumbelliferyl-beta-D-glucuronide ester (4-MUG) as a substrate, and GUS enzyme catalyzes the hydrolysis of the substrate to obtain 4-methylumbelliferone (4-MU) and beta-D glucuronic acid. The hydroxyl in the 4-MU molecule is dissociated and then excited by 365nm light to generate 455nm fluorescence, which can be quantified by a fluorescence spectrophotometer.
5.2.1 GUS crude protein extraction
Grinding about 0.1g tobacco leaf tissue into powder with liquid nitrogen, transferring into a 1.5mL centrifuge tube precooled by liquid nitrogen, adding 200 μ L GUS fluorescent quantitative extracting solution, thawing at room temperature, mixing uniformly, and centrifuging at 12000r/min for 15 min. Transferring the supernatant into a new centrifuge tube, subpackaging, and storing at-70 ℃ for later use.
5.2.2 preparation of Standard Curve
Bovine serum albumin BSA 25mg was weighed accurately, dissolved in water and made to volume of 100 mL. The solution is sucked up by 40mL, diluted by distilled water to 100mL, and the volume is the standard protein solution of 100 mug/mL. 100mg of Coomassie brilliant blue G250 is weighed, dissolved in 50mL of 90% ethanol, 100mL of 85% (W/V) phosphoric acid is added, the volume is adjusted to 1000mL by distilled water, and the mixture is filtered by filter paper and stored in dark at 4 ℃. 6 tubes of 10mL were numbered and the reagents were added as in the following table (Table 8).
TABLE 8 Standard Curve for protein concentration
Figure BDA0003132904860000111
Mixing, and making the oscillation degree of each tube consistent as much as possible. Standing for 10min, and performing colorimetric determination at 595 nm. A standard curve was plotted with bovine serum albumin (μ g) as the abscissa and the pipette value as the ordinate.
5.2.3 determination of protein content in samples
Sucking 50 μ L GUS crude protein extract, adding 950 μ L ddH2O, then adding 5mL of G250 solution, mixing uniformly, standing at room temperature for 10min, and then measuring the absorbance at 595 nm. Protein concentrations were calculated from the standard curve.
5.2.4 GUS fluorometric assay
(1) And (3) preparing a standard curve: 0.2032g of 4-MUG is accurately weighed, added with water, dissolved to 100mL, and prepared into solution with the concentration of 5mmol/L (table 9).
TABLE 9 preparation of MUG Standard Curve
Figure BDA0003132904860000112
GUS fluorescence assay: mixing 70 μ L GUS crude protein extract and 80 μ L GUS reaction solution, immediately sucking 30 μ L liquid to 60 μ L0.2 mol/L Na2CO3The reaction was terminated. The rest reaction solution is put in water bath at 37 ℃, and 240 mu L of 0.2mol/L Na is added after 1 hour2CO3The reaction was terminated, and after mixing, 90. mu.L of the mixture was pipetted out of the wells and read in a 96-well microplate. Values of 0h and 1h were read using a Millipore luminescence detector at excitation 340nm and emission 465 nm.
Calculation of GUS Activity: the 4-MU concentration at 0h and 1h was calculated according to the standard curve, the GUS activity value was 1h of 4-MU concentration minus 0h of 4-MU concentration, divided by 1h (warm bath time) and protein concentration in nmol 4-MU/mg/min.
6. Functional validation of ZmOMAp1730 sequences
The qRT-PCR results show that the ZmOMA gene promoter responds to drought stress. Moreover, according to the distribution of cis-acting elements in the ZmOMA gene promoter, GUS histochemical staining and GUS protein quantification results of GUS expression vectors of promoters with different lengths and GUS expression vectors of full-length promoters show that the GUS activity of the GUS expression vector started by the promoter with 1730bp is the highest. Therefore, 1730bp of sequence was selected for its subsequent study and named ZmOMAp 1730.
6.1 functional verification of ZmOMAp1730 sequence expression vector in tobacco
Transforming agrobacterium with the constructed P1730-GUS expression vector, injecting the agrobacterium into tobacco leaves by the same method, culturing the tobacco plants after injection at 25 ℃ for 24h under 14/10h photoperiod conditions, carrying out 20% PEG simulated drought treatment on the tobacco plants, and taking the leaves for GUS histochemical staining and GUS activity quantitative analysis at the treatment time points of 0, 3, 6, 12 and 24 h.
6.2 functional verification of ZmOMAp1730 sequence expression vector in Arabidopsis thaliana
Transforming the constructed P1730-GUS expression vector into agrobacterium, transforming wild arabidopsis thaliana by a floral infection method, and screening transgenic positive seedlings until obtaining T3 generation homozygous transgenic seeds. Will T3And culturing the generation homozygous transgenic plant strain for 14 days at 25 ℃ under 14/10h photoperiod, performing 20% PEG simulated drought treatment on the generation homozygous transgenic plant strain, and taking transgenic arabidopsis thaliana plants for GUS histochemical staining and GUS activity quantitative analysis at the treatment time points of 0, 3, 6, 12 and 24 h.
7. Analysis of results
7.1 expression Pattern analysis of ZmOMA Gene
Maize C01 inbred plants were subjected to 0, 3, 6, 12, 24h 20% PEG treatment. And detecting the gene expression quantity of the treated plants at different positions and different treatment times.
The result shows that the expression level of the gene is not obviously changed in the early stages of 0, 3 and 6 hours of treatment in the corn leaves, the expression level of the gene is increased after 12 hours of treatment, and the expression level of the gene is obviously increased after 24 hours of treatment; the expression rule of the gene in the corn root is about the same as that in the leaf, and the expression amount of the gene in the corn root is very different after 12h and 24h of treatment (figure 1), and the ZmOMA gene promoter is suspected to respond to drought stress.
7.2 ZmOMA Gene promoter sequence analysis and primer design
According to plantarcae
(http:// bioinformatics. psb. content. be/webtools/plantare/html /) website analysis ZmOMA gene promoter sequence result shows that the 2000bp promoter sequence contains a large amount of cis-acting elements related to drought response, such as MBS, MYB, MYC, Myb and the like (figure 2), and the promoter is conjectured to be related to drought stress by combining the previous qRT-PCR result, so that primers are designed according to the positions of drought response elements in the promoter (figure 2) to amplify ZmOMA gene promoter 5' end deletion sequences with different lengths.
7.3 amplification of the ZmOMA Gene promoter
The corn genomic DNA was extracted and the corresponding promoter fragment was amplified based on the amplification primer of ZmOMA gene promoter (FIG. 3). Recovering the amplified fragments, and connecting with vector pRI201-AN-GUS to obtain 6 GUS expression vectors (P)2000、P1730、P1430、P1150、P745、P100)。
7.4 the ZmOMAp1730 promoter has the highest activity
Six GUS expression vectors P constructed2000、P1730、P1430、P1150、P745、P100Transient expression is carried out by injecting tobacco through an agrobacterium-mediated method, GUS histochemical staining and GUS quantitative analysis show that: and P2000In contrast, only P1730The expression level was significantly increased (fig. 4). The promoter fragment is guessed to contain a key region responding to drought stress, so that a P1730-GUS expression vector is selected to carry out a subsequent drought verification experiment and is named as ZmOMAp 1730.
7.5 ZmOMAp1730 Start Activity induced by drought
The P1730-GUS expression vector is injected into tobacco by an agrobacterium-mediated method for transient expression. GUS histochemical staining and GUS quantitative analysis show that: as the drought treatment time was prolonged, the expression level of GUS protein was increased (FIG. 5).
In order to further verify that the ZmOMAp1730 promoter responds to drought stress, the P1730-GUS expression vector is used for infecting arabidopsis thaliana by a floral flocculation infection method for stable expression. GUS histochemical staining and GUS quantitative analysis show that: as the drought treatment time was prolonged, the expression level of GUS protein was increased (FIG. 6).
By comparison of promoter fragments of different lengths with the full-length promoter, where P1730The GUS expression vector has the highest expression level. In addition, further drought treatment experiments (transient expression and stable expression) showed that P1730As the drought time is prolonged, the expression level of the GUS expression vector is increased. The research results prove that the ZmOMAp1730 gene promoter responds to drought stress and is positively regulated by the drought stress.
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
Sequence listing
<110> Sichuan university of agriculture
<120> corn drought inducible promoter ZmOMAp1730 and application thereof
<160> 9
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1730
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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attggtttgt ctgcctctgt tgtgagtttg ttcctgcgtg gtaaaaatac gtaccttgtt 60
attggtagat ggctcgttga tggagtgcat tggattggtg gtcctcctga agactacatt 120
gctgactggt tgtgtcattg tgtgtgatga gagatcttcc agaacttgtg ctgaagtata 180
tgttcctgca taagacaaat tagagttttg atcaggtagg tgcttacccc caaggttttg 240
tgtccattgg tttcttcaga atgagctgcg ttgttgactg ctatatgtgc ggctattttc 300
attatgaact tgcacaactg tccaagattt tggaattgaa tctcttgtcg tttctggctt 360
tccaaatgta ccgaatcatc aggagtatat ggttggatca gagctgcaaa aatttcctgg 420
actccatctt gttcctgagg gaggttagta gtacgtagaa gagggataga agaaaatgag 480
taaaatgtat cagcactaat tgaacttgac atgttgtgtc acttggatct tgaacttaaa 540
aaaacagaaa aacaggtcct aaactaaaaa acaggaaaac atgtcttaaa cttgatggac 600
ctatgcaaaa tcatccaaaa attggatatc aggatatgca tcattttcca gcacgttcgc 660
ctgtggctaa aggctaaggc tgtctccagc aacgtactct aaatttcatc ccctaaagga 720
atattctttg tcctttacag cacactctaa aagattacat cctctatatc ttcgtcatct 780
ccaacaacgt cctctaaatt tcatcctcta tatctcatcg ttcgtatttt caccgaccac 840
tattatcaga aaattttaaa tctatattta tccacacatt tttaactaat ttaaatatta 900
caaattgttt ttgtatttat attaaaaaat atgttttaga actataactt ttttaccatg 960
cgcaaatatt ttttatctaa acaaatctct aaatcccaca tatcccatcc tcttcccagg 1020
cgtttcccac tcccgagcag acgctgctag cacggtgttc atctttgatt tgagctcacc 1080
gctgcgcagc aattgtgggc cgccattcgt gtggacagag agtggtggga acgagaatgg 1140
ttggccgagg tcctacgcgc cgctgcggcg ctcgagcacg gctccgacga tgagatggat 1200
ggcgcggccg aggccctccg tgccgccaca tccccaatcc tctgcatcca cttttcgctc 1260
ctctccggct cctctccggc gagcgaatcg ccgtggatct cgatgagaag gatgctggag 1320
catggcacga acgcggatag cgaacgcaag ctcgtccgct aggattaccg aacgatgagg 1380
accctgtaaa atttagcgca tcttttagcg tctattgctg gagacgtaaa gtaagtcacc 1440
gttcgctaca tttaggggag aggacccttt acagggtgtt gctggagaca gcctaaagct 1500
ctccaggcga aggttttgat gtgcttccag gtgcgttgta gtatctgcac tgctgctgac 1560
atgttgagtc ctgtactccg tcatatatgg gcctacgata cgaaactggg ccttgcgaag 1620
tttcccagca agtgctagag agtgccaggc gacacggttc gctcggcggc ggcgccatct 1680
ggcagcttct gcaaaactga agtgcccatt ccagccctcc cgtagcgatc 1730
<210> 2
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
acgacggcca gtgccaagct tattggtttg tctgcctctg tt 42
<210> 3
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
tgtgattgtg atgtatctag agatcgctac gggagggctg 40
<210> 4
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
acgacggcca gtgccaagct ttaagatcaa aattcaaccg t 41
<210> 5
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
acgacggcca gtgccaagct tattatgaac ttgcacaact g 41
<210> 6
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
acgacggcca gtgccaagct tatgtcttaa acttgatgga c 41
<210> 7
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
acgacggcca gtgccaagct ttctctaaat cccacatatc cc 42
<210> 8
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
acgacggcca gtgccaagct tagtgctaga gagtgccagg c 41
<210> 9
<211> 12220
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc 60
cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga 120
aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct 180
cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg 240
gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag 300
ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat 360
cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac 420
aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac 480
tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc 540
ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt 600
tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc 660
ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg 720
agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca 780
atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca 840
cctatctcag cgatctgtct atttcgttca tccatagttg cctgactcga tcctacaagg 900
tagaatccgc ctgagtcgca agggtgactt cgcctatatt ggacgacggc gcgcagaggg 960
cgacctcttt ttgggttacg attgtaggat tatcactaaa acaatacatg aacatattca 1020
aatggcaatc tctctaaggc attggaaata aatacaaata acagttgggt ggagtttttc 1080
gacctgaggg cgttaacctt ctgttaacct aaaagctctt gcccaaacag cagaatcggc 1140
gctaattgcc agcggcggaa cttttccagt ttcgcgaaaa atatcgccac tggcaaggaa 1200
tgggtttgag atggcgaagt ctgtcctaaa agcagcgcct gtagttgtag ggttgacggc 1260
cttgatggag cgtcatgccg atgccctctc gagccaactt caagcacatc atcttaaggt 1320
tttcccgccg cattccgaga agggtattcg aacattcggg ccatcggagg cgtccaagct 1380
gctcggcgtt ggcgagtcat atttacggca gaccgcgtct gagatgccag agttgaatgt 1440
tagcatgagc ccaggtggca ggcgaatgtt ctcaattgaa gatatccatg tgattcggaa 1500
gtatatggat caggtcggcc gcgggaaccg gcgctacctg ccacatcgtc gaggcggcga 1560
gcagcttcag gttatctctg tgatgaattt caaaggtggg tcgggtaaga ccaccaccgc 1620
cgcgcatctg gcgcagtacc tcgctatgcg cggatatcga gtcttggcca ttgatctcga 1680
tcctcaagcg agcctttctg cactctttgg gagccaaccg gagacggacg ttggcccgaa 1740
cgaaacgctc tacggcgcta taaggtatga tgatgagcag gtggcaatcg aacgagtcgt 1800
ccgagggact tacattcccg acctccacct gattcctggt aaccttgagc tgatggagtt 1860
tgaacacgat acgccacgcg cgctgatgaa ccgcaaagag ggcgacacgc tcttttatgg 1920
tcgcatcagc caagtaattg aagatatcgc ggataactat gacgtcgtgg tcatcgactg 1980
ccctccccag cttgggtatc tcacgctatc cgcattgact gcggcgacgt ccattcttgt 2040
cacggtccat ccgcagatgc tggatgtgat gtcgatgaac cagtttctgg caatgacatc 2100
gaaccttttg cgtgaaatcg agaatgctgg cgccaagttc aagtttaatt ggatgcgcta 2160
tctgataacc cgtttcgaac cgagcgacgg accacagaac caaatggtag gttatctgcg 2220
gtcgattttt ggcgaaaatg tcctcaattt tccgatgctt aaaaccaccg cggtttcgga 2280
cgctggcctg acaaaccaga ctctattcga agtggagcgt ggcctgttca cgcgctcgac 2340
ctatgatcga gccttggagg cgatgaacgc cgtcaacgac gagatcgaaa cactgatcaa 2400
aaaagcatgg ggtaggccca catgagccgg aagcacatcc ttggcgtctc aactgacgcc 2460
cctgagacgt cgcccgccga caataggacg gcaaagaacc gctccatgcc gctcctcggc 2520
gtaacaagga aggagcgcga tccggcaacg aagctcacag cgaacattgg taacgcactg 2580
cgagagcaaa acgatcgtct tagccgtgcc gaagagatcg agcggcgtct cgctgaaggt 2640
caggcagtga tagagttgga tgcctcgtca atagaaccgt ctttcgtgca ggatcgtatg 2700
cgaggggaca ttgacgggct ccttacttcg atccgggaac aaggacagca agtcccaatc 2760
cttgtgcgac cgcatccgag ccagccgggc cgatatcagg ttgccttcgg ccaccgccgg 2820
ctacgcgccg tttcagaact cggacttccg gtcagggcgg tcgttcgcga actgacggac 2880
gagcaagtgg tcgtagcaca gggtcaggaa aacaatgtgc gcgaagatct taccttcatc 2940
gaaaaggcgc gcttcgcaca tcgcctgaac aggcagtttt ctcgagagat tgtcatcgcc 3000
gcgatgtcga tcgacaagag caatttgtcc aagatgcttc tgctcgttga cgccctcccc 3060
tctgaactga ccgatgctat tggtgccgct cctggtgttg gacggccgag ttggcaacaa 3120
cttgccgagc tgattgagaa agtttcttca ccggccgacg tggctaaata tgctatgtcg 3180
gaggaagttc aagcgctgcc atcggcagaa cgattcaagg cggtgatcgc tagtctgaag 3240
cccagtcggg ttgcgcgtgg acttcccgag gtcatggcca ccccagacgg caccagaatt 3300
gcacaggtga cgcagagcaa ggccaaactg gaaatcacga ttgacaggaa ggcgacgccc 3360
gattttgcga ccttcgtgct cgatcatgtg ccagcgctgt atcaagcgta ccacgctgag 3420
aaccaacgga aacggggaga gtaaaccgca aaagaaaaga gccccctcaa cgtcgccgtc 3480
gcggaagccc ttctgtctct ctagcgcgaa cagaatcgca tttcctcgaa tcctcgtcaa 3540
gagtttttag cgccgttttg gtgagctgat ttcctttgcc tgctgaaagg tgaaagatga 3600
tgcagacagg aagtgtaacg acgccattcg ggcggcggcc aatgacgctt gcgcttgtgc 3660
ggcgccagac ggcgctggcc gatatcaaac aaggcaagac agcggacaag tggaaggtct 3720
ttagggacgc gtccgcggcc atggaactac ttggaatcca gtccaacagt cttgccgtcc 3780
ttgatgcgct attgagcttt cacccggaaa cggagttgcg tcaggaggca cagctgatcg 3840
tcttcccgtc gaatgctcag cttgcccttc gggcgcatgg gatggctggc gcgactttgc 3900
gtaggcacat cgccatgctc gtggagtcag gcttgatcgt ccggaaggat agcgccaacg 3960
gaaagcgtta cgctcgtaag gatggcgctg gtcagatcga gcgcgcgttt ggcttcgatt 4020
tgtctccgct tctcgcgcgg tccgaagagc tagcgatgat ggcacagcag gtgatggccg 4080
atcgagcagc attcaggatg gccaaagaaa gtctgacgat ttgccgacgg gacgttcgga 4140
agctaattac ggcagctatg gaagagggag cggagggcga ctggcaagct gtcgaggaag 4200
tctatgtgga acttgtgggt agaatcccac gcgccccgac gcttgctgat gtagagtcaa 4260
ttctcgaaga gatgtggatg ctccaggaag agataatcaa ccggttggaa attagagaca 4320
attcagaaaa taatagcacc aatgctgccc agagcgagca gcacatacag aactcaaaac 4380
ccgaatccgt taatgaactt gaacctcgct ctgaaaagga gcagggcgct aagccgagtg 4440
aaatagaccg ggcaaggagc gagccgataa aagcgttccc cctcgggatg atcctgaaag 4500
cgtgcccgac cattggcaat tatgggccga gcggtgcggt tgctagctgg cgtgacctca 4560
tgtcggctgc ggtggtggtt cggtctatgc tgggggtcag cccgtcggct taccaagacg 4620
cgtgtgaggc aatgggaccg gagaatgcgg cagcagcgat ggcgtgcatt ttggagcgag 4680
cgaacttcat caattcgcct gggggctatc tccgagatct gacacggcgg agcgaactcg 4740
ggaagttttc acttggcccg atgataatgg cgctcttgaa ggctagcggg caggggacgt 4800
tgcggtttgg ctagaattag cgagtatgga gcaggatggt ctgtggtcag ctgaccacag 4860
acctaatagg ttgaaaacat gagcgttttt tggatgatcg acagaccatc cgattcccgg 4920
agtaccaagc gtgctctgat gggagcgata acattactca acaagcacga aggccccatg 4980
ccgatcgttg atcgtgaagg agagcctgct ctacatgcgg cggtattttg ccggccgagg 5040
catgtagtcg cggagcactg cctatttact gccctaggca caaacgttga ctcttggatc 5100
gagctggcag acaaagcaat aacccacaca gaggacgatt aatggctgac gaagagatcc 5160
agaatccgcc ggacggtact gctgctgccg aagttgagcc ggctgctcct agaggtagaa 5220
gagcaaagaa agcaccagcc gaaacagccc gcacgggatc gttcaaatcc gtgaagccga 5280
aaacccgcgg cctcagcaac cgagaaaaac tggagaagat cggtcaaatc gaagctcagg 5340
tcgctggcgg cgcaaccttg aaggacgccg ttaagatcgt gggtatttcc gttcagacct 5400
attatcaatg gaagagagct gcggttcaac ctgtctcaca gaatccggcc gtgtctgttt 5460
cagttgacga tgaactcggc gagttcatcc aactcgagga ggaaaatcgg cggctcagaa 5520
agctagcttg tggcaggata tattgtggtg taaacaaatt gacgcttaga caacttaata 5580
acacattgcg gacgttttta atgtactggg gtggtttttc ttttcaccag tgagacgggc 5640
aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg 5700
gtttgcccca gcaggcgaaa atcctgtttg atggtggttc cgaaatcggc aaaatccctt 5760
ataaatcaaa agaatagccc gagatagggt tgagtgttgt tccagtttgg aacaagagtc 5820
cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg 5880
gcccaaattc ccgatctagt aacatagatg acaccgcgcg cgataattta tcctagtttg 5940
cgcgctatat tttgttttct atcgcgtatt aaatgtataa ttgcgggact ctaatcataa 6000
aaacccatct cataaataac gtcatgcatt acatgttaat tattacatgc ttaacgtaat 6060
tcaacagaaa ttatatgata atcatcgcaa gaccggcaac aggattcaat cttaagaaac 6120
tttattgcca aatgtttgaa cgatcggggc ccgatcatat tgtcgctcag gatcgtggcg 6180
ttgtgcttgt cggccgttgc tgtcgtaatg atatcggcac cttcgaccgc ctgttccgca 6240
gagatcccgt gggcgaagaa ctccagcatg agatccccgc gctggaggat catccagccg 6300
gcgtcccgga aaacgattcc gaagcccaac ctttcataga aggcggcggt ggaatcgaaa 6360
tctcgtgatg gcaggttggg cgtcgcttgg tcggtcattt cgaaccccag agtcccgctc 6420
agaagaactc gtcaagaagg cgatagaagg cgatgcgctg cgaatcggga gcggcgatac 6480
cgtaaagcac gaggaagcgg tcagcccatt cgccgccaag ctcttcagca atatcacggg 6540
tagccaacgc tatgtcctga tagcggtccg ccacacccag ccggccacag tcgatgaatc 6600
cagaaaagcg gccattttcc accatgatat tcggcaagca ggcatcgcca tgggtcacga 6660
cgagatcatc gccgtcgggc atacgcgcct tgagcctggc gaacagttcg gctggcgcga 6720
gcccctgatg ctcttcgtcc agatcatcct gatcgacaag accggcttcc atccgagtac 6780
gtgctcgctc gatgcgatgt ttcgcttggt ggtcgaatgg gcaggtagcc ggatcaagcg 6840
tatgcagccg ccgcattgca tcagccatga tggatacttt ctcggcagga gcaaggtgag 6900
atgacaggag atcctgcccc ggcacttcgc ccaatagcag ccagtccctt cccgcttcag 6960
tgacaacgtc gagcacagct gcgcaaggaa cgcccgtcgt ggccagccac gatagccgcg 7020
ctgcctcgtc ctgtagttca ttcagggcac cggacaggtc ggtcttgaca aaaagaaccg 7080
ggcgcccctg cgctgacagc cggaacacgg cggcatcaga gcagccgatt gtctgttgtg 7140
cccagtcata gccgaatagc ctctccaccc aagcggccgg agaacctgcg tgcaatccat 7200
cttgttcaat catgcgaaac gatccagatc cggtgcagat tatttggatt gagagtgaat 7260
atgagactct aattggatac cgaggggaat ttatggaacg tcagtggagc atttttgaca 7320
agaaatattt gctagctgat agtgacctta ggcgactttt gaacgcgcaa taatggtttc 7380
tgacgtatgt gcttagctca ttaaactcca gaaacccgcg gctgagtggc tccttcaacg 7440
ttgcggttct gtcagttcca aacgtaaaac ggcttgtccc gcgtcatcgg cgggggtcat 7500
aacgtgactc ccttaattct ccgctcatga tcctggcacg acaggtttcc cgactggaaa 7560
gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct 7620
ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac 7680
acaggaaaca gctatgacca tgattacgaa ttcccgggat ccagcggccg caggtaccaa 7740
ttccttatct ttaatcatat tccatagtcc ataccatagc acatacagta gttatatgcg 7800
aagagatcca acaaaacatt cacaatggat tatagaaaca tttgtttatt cattataatg 7860
agatcttaca ttcatttaat attagaaaaa gccacaaatt cataacacaa caagccaaga 7920
aaaaaacaca aacttaagca cacaagctag ctttttattt gacacaccaa atatttcatc 7980
ttcatcttca taagagctcg gtagcaattc ccgaggctgt agccgacgat ggtgcgccag 8040
gagagttgtt gattcagaat tgtcgaggtc gatcattgtt tgcctccctg ctgcggtttt 8100
tcaccgaagt tcatgccagt ccagcgtttt tgcagcagaa aagccgccga cttcggtttg 8160
cggtcgcgag tgaagatccc tttcttgtta ccgccaacgc gcaatatgcc ttgcgaggtc 8220
gcaaaatcgg cgaaattcca tacctgttca ccgacgacgg cgctgacgcg atcaaagacg 8280
cggtgataca tatccagcca tgcacactga tactcttcac tccacatgtc ggtgtacatt 8340
gagtgcagcc cggctaacgt atccacgccg tattcggtga tgataatcgg ctgatgcagt 8400
ttctcctgcc aggccagaag ttctttttcc agtaccttct ctgccgtttc caaatcgccg 8460
ctttggacat accatccgta ataacggttc aggcacagca catcaaagag atcgctgatg 8520
gtatcggtgt gagcgtcgca gaacattaca ttgacgcagg tgatcggacg cgtcgggtcg 8580
agtttacgcg ttgcttccgc cagtggcgcg aaatattccc gtgcaccttg cggacgggta 8640
tccggttcgt tggcaatact ccacatcacc acgcttgggt ggtttttgtc acgcgctatc 8700
agctctttaa tcgcctgtaa gtgcgcttgc tgagtttccc cgttgactgc ctcttcgctg 8760
tacagttctt tcggcttgtt gcccgcttcg aaaccaatgc ctaaagagag gttaaagccg 8820
acagcagcag tttcatcaat caccacgatg ccatgttcat ctgcccagtc gagcatctct 8880
tcagcgtaag ggtaatgcga ggtacggtag gagttggccc caatccagtc cattaatgcg 8940
tggtcgtgca ccatcagcac gttatcgaat cctttgccac gcaagtccgc atcttcatga 9000
cgaccaaagc cagtaaagta gaacggtttg tggttaatca ggaactgttc gcccttcact 9060
gccactgacc ggatgccgac gcgaagcggg tagatatcac actctgtctg gcttttggct 9120
gtgacgcaca gttcatagag ataaccttca cccggttgcc agaggtgcgg attcaccact 9180
tgcaaagtcc cgctagtgcc ttgtccagtt gcaaccacct gttgatccgc atcacgcagt 9240
tcaacgctga catcaccatt ggccaccacc tgccagtcaa cagacgcgtg gttacagtct 9300
tgcgcgacat gcgtcaccac ggtgatatcg tccacccagg tgttcggcgt ggtgtagagc 9360
attacgctgc gatggatccc ggcatagtta aagaaatcat ggaagtaaga ctgctttttc 9420
ttgccgtttt cgtcggtaat caccattccc ggcgggatag tctgccagtt cagttcgttg 9480
ttcacacaaa cggtgatacg tacacttttc ccggcaataa catacggcgt gacatcggct 9540
tcaaatggcg tatagccgcc ctgatgctcc atcacttcct gattattgac ccacactttg 9600
ccgtaatgag tgaccgcatc gaaacgcagc acgatacgct ggcctgccca acctttcggt 9660
ataaagactt cgcgctgata ccagacgttg cccgcataat tacgaatatc tgcatcggcg 9720
aactgatcgt taaaactgcc tggcacagca attgcccggc tttcttgtaa cgcgctttcc 9780
caccaacgct gatcaattcc acagttttcg cgatccagac tgaatgccca caggccgtcg 9840
agttttttga tttcacgggt tggggtttct acaggacgta acatatgtat caacagtgaa 9900
gaacttgctt ttgatctttt gttagttttg tgtgattgtg atgtatctag agtcccccgt 9960
gttctctcca aatgaaatga acttccttat atagaggaag ggtcttgcga aggatagtgg 10020
gattgtgcgt catcccttac gtcagtggag atatcacatc aatccacttg ctttgaagac 10080
gtggttggaa cgtcttcttt ttccacgatg ctcctcgtgg gtgggggtcc atctttggga 10140
ccactgtcgg cagaggcatc ttcaacgatg gcctttcctt tatcgcaatg atggcatttg 10200
taggagccac cttccttttc cactatcttc acaataaagt gacagatagc tgggcaatgg 10260
aatccgagga ggtttccgga tattaccctt tgttgaaaag tctcaattgc cctttggtct 10320
tctgagactg tatctttgat atttttggag tagacaagtg tgtcgtgctc caccatgttg 10380
acgaagattt tcttcttgtc attgagtcgt aagagactct gtatgaactg ttcgccagtc 10440
tttacggcga gttctgttag gtcctctatt tgaatctttg actccatggc ctttgattca 10500
gtgggaacta cctttttaga gactccaatc tctattactt gccttggttt gtgaagcaag 10560
ccttgaatcg tccatactgg aatagtactt ctgatcttga gaaatatatc tttctctgtg 10620
ttcttgatgc agttagtcct gaatcttttg actgcatctt taaccttctt gggaaggtat 10680
ttgatttcct ggagattatt gctcgggtag atcgtcttga tgagacctgc tgcgtaagcc 10740
tctctaacca tctgtgggtt agcattcttt ctgaaattga aaaggctaat ctggggacct 10800
gcaggcatgc aagcttggca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg 10860
gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccaggatc agattgtcgt 10920
ttcccgcctt cagtttaaac tatcagtgtt tgacaggata tattggcggg taaacctaag 10980
agaaaagagc gtttattaga ataatcggat atttaaaagg gcgtgaaaag gtttatccgt 11040
tcgtccattt gtatgtgact aaaacaattc atccagtaaa atataatatt ttattttctc 11100
ccaatcaggc ttgatcccca gtaagtcaaa aaatagctcg acatactgtt cttccccgat 11160
atcctccctg atcgaccgga cgcagaaggc aatgtcatac cacttgtccg ccctgccgct 11220
tctcccaaga tcaataaagc cacttacttt gccatctttc acaaagatgt tgctgtctcc 11280
caggtcgccg tgggaaaaga caagttcctc ttcgggcttt tccgtcttta aaaaatcata 11340
cagctcgcgc ggatctttaa atggagtgtc ttcttcccag ttttcgcaat ccacatcggc 11400
cagatcgtta ttcagtaagt aatccaattc ggctaagcgg ctgtctaagc tattcgtata 11460
gggacaatcc gatatgtcga tggagtgaaa gagcctgatg cactccgcat acagctcgat 11520
aatcttttca gggctttgtt catcttcata ctcttccgag caaaggacgc catcggcctc 11580
actcatgagc agattgctcc agccatcatg ccgttcaaag tgcaggacct ttggaacagg 11640
cagctttcct tccagccata gcatcatgtc cttttcccgt tccacatcat aggtggtccc 11700
tttataccgg ctgtccgtca tttttaaata taggttttca ttttctccca ccagcttata 11760
taccttagca ggagacattc cttccgtatc ttttacgcag cggtattttt cgatcagttt 11820
tttcaattcc ggtgatattc tcattttagc catttattat ttccttcctc ttttctacag 11880
tatttaaaga taccccaaga agctaattat aacaagacga actccaattc actgttcctt 11940
gcattctaaa accttaaata ccagaaaaca gctttttcaa agttgttttc aaagttggcg 12000
tataacatag tatcgacgga gccgattttg aaaccacaat tatgggtgat gctgccaact 12060
tactgattta gtgtatgatg gtgtttttga ggtgctccag tggcttctgt ttctatcagc 12120
tgtccctcct gttcagctac tgacggggtg gtgcgtaacg gcaaaagcac cgccggacat 12180
cagcgctatc tctgctctca ctgccgtaaa acatggcaac 12220

Claims (5)

1. An obtained corn drought inducible promoter ZmOMAp1730 is characterized in that the nucleotide sequence of the promoter ZmOMAp1730 is shown as SEQ ID NO: 1, 1-1730 th base sequence.
2. A primer for amplifying the maize drought inducible promoter ZmOMAp1730 of claim 1, wherein the nucleotide sequence is as shown in SEQ ID NO: 2 and the upstream primer shown in SEQ ID NO: 3 as shown in the figure.
3. The primer of claim 2, wherein the amplification procedure is pre-denaturation at 94 ℃ for 3 min; denaturation at 98 ℃ for 10s, annealing at 68 ℃ for 10s, extension at 72 ℃ for 2min, and 30 cycles; further extension at 72 deg.C for 5 min; keeping at 4 ℃.
4. The application of the maize drought-inducible promoter ZmOMAp1730 as described in claim 1, which is applied in the following (a) or (b):
(a) the application in regulating and controlling plant drought stress;
(b) application in plant drought-resistant breeding.
5. The use of claim 4, wherein the plant comprises maize, tobacco and Arabidopsis.
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