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. Use of the maize drought-inducible promoter ZmOMAp1730 as defined in claim 1 or the primers as defined in any of claims 2 to 3 in (a) or (b) as follows:

(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.

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.

Drawings

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 PrimeScript^TMThe 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). Fluorescent staining with SYBGreenThe material was subjected to qRT-PCR, the reaction system according to Table 2 was loaded, CFX96^TMAmplification 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

TABLE 2 qRT-PCR reaction System

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

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

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

TABLE 6 ligation reaction System

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) Adding 10mL of precooled 0.15mol/L NaCl solution, suspending the cells, and 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)

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

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

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

Extracting maize genomic DNA from ZmOMA gene promoterThe corresponding promoter fragment was amplified by the amplification primer of (1) (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

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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