Promoter of bombyx mori pebrine induced expression gene BmPGT 3 and application thereof

文档序号：758698 发布日期：2021-04-06 浏览：24次中文

阅读说明：本技术 家蚕微粒子虫诱导表达基因BmPUGT3的启动子及其应用 (Promoter of bombyx mori pebrine induced expression gene BmPGT 3 and application thereof ) 是由李春峰于滨潘国庆周泽扬于 2020-12-10 设计创作，主要内容包括：本发明属于家蚕转基因技术领域,具体涉及一种家蚕微粒子虫诱导表达基因BmPUGT3的启动子及其应用,所述启动子的核苷酸基因如SEQ ID NO：16所示,该启动子可以驱动外源基因在家蚕中经家蚕微粒子虫诱导下表达,不但适用于基因功能分析等分子生物学理论研究,同时适用于利用基因工程进行家蚕品种改良,特别是家蚕抗微粒子病的品种培育,具有良好的应用前景。(The invention belongs to the technical field of silkworm transgenosis, and particularly relates to a promoter of a bombyx mori nosema induced expression gene BmPGT 3 and application thereof, wherein a nucleotide gene of the promoter is shown as SEQ ID NO: 16, the promoter can drive the expression of exogenous genes in silkworms under the induction of silkworm nosema bombycis, is not only suitable for molecular biology theory research such as gene function analysis, but also suitable for improving the silkworm varieties by using genetic engineering, particularly for breeding the silkworm nosema bombycis anti-disease varieties, and has good application prospect.)

1. A promoter of bombyx mori pebrine induced expression gene BmPGT 3 is characterized in that the nucleotide sequence of the promoter is shown as SEQ ID NO: shown at 16.

2. A recombinant expression vector comprising the promoter of claim 1.

3. The method for constructing the recombinant expression vector of claim 2, wherein the method comprises designing upstream and downstream primers containing the restriction enzyme sites of the expression vector according to the nucleotide sequence of the promoter of claim 1, performing PCR amplification by using silkworm genome DNA as a template to obtain a target fragment containing the promoter, and connecting the target fragment to the expression vector to obtain the recombinant expression vector.

4. The method of claim 3, wherein the expression vector is the pSLfa1180fa vector.

5. The method of claim 3 or 4, wherein the nucleotide sequence of the forward primer is as set forth in SEQ ID NO: 17, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 18, respectively.

6. A recombinant cell transformed with the recombinant expression vector of claim 2 into a recipient cell.

7. A foreign protein expressed by the recombinant cell of claim 6.

8. The use of the promoter of claim 1 for breeding bombyx mori resistant to microparticulate insects.

Technical Field

The invention belongs to the technical field of silkworm transgenosis, and particularly relates to a promoter of a bombyx mori nosema induced expression gene BmPGT 3 and application thereof.

Background

The silk industry is the traditional dominant industry in China, still dominates the international market so far, and is an important economic income source for tens of millions of farmers. Silkworm pebrine is a head blight which jeopardizes the safety production of silkworm industry, and the fundamental reason is that the pathogen of the disease, namely silkworm pebrine (Nosema bombycis), can vertically spread through eggs to cause silkworm virus. Currently, the direct economic loss of China due to the microparticulate disease reaches billions of yuan every year, and the cost of the main silkworm breeding farms in China for preventing and controlling the disease accounts for half of the total cost of silkworm breeding. However, silkworm material resistant to the disease has not been found in the production at present.

With the breakthrough and improvement of the transgenic technology of silkworms, the transgenic technology has become a powerful tool for breeding resistant materials of silkworms. However, constitutive expression promoters are still used in breeding of transgenic silkworms. The constitutive promoter is utilized, on one hand, the expression efficiency of the exogenous gene is reduced, and on the other hand, the constitutive promoter can bring burden to the silkworm. At present, there are few reports about the Bombyx mori inducible promoter. Therefore, the development of the bombyx mori inducible promoter has great significance for reducing the load of exogenous genome constitutive expression in the construction of bombyx mori resistance breeding materials, improving the resistance of transgenic bombyx mori and developing a new bombyx resistance system.

Disclosure of Invention

In view of the above, an object of the present invention is to provide a promoter of bombyx mori pebrine inducible expression gene bmpgt 3, a second object is to provide a recombinant expression vector, a third object is to provide a construction method of the recombinant expression vector, a fourth object is to provide a recombinant cell, a fifth object is to provide a foreign protein, and a sixth object is to provide an application of the promoter of bombyx mori pebrine inducible expression gene bmpgt 3 in breeding of bombyx mori resistant bombyx mori.

In order to achieve the purpose, the invention provides the following technical scheme:

1. a promoter of bombyx mori pebrine induced expression gene BmPGT 3, wherein the nucleotide sequence of the promoter is shown as SEQ ID NO: shown at 16.

2. A recombinant expression vector comprises a promoter of the bombyx mori nosema bombycis inducible expression gene BmPGT 3.

3. A construction method of a recombinant expression vector comprises the steps of designing upstream and downstream primers containing enzyme cutting sites of the expression vector according to a nucleotide sequence of a promoter of a bombyx mori micro-particle worm induced expression gene BmPGT 3, carrying out PCR amplification by taking bombyx mori genome DNA as a template to obtain a target fragment containing the promoter, and connecting the target fragment to the expression vector to obtain the recombinant expression vector.

As one of the preferable technical schemes, the expression vector is pSLfa1180fa vector.

As one of the preferable technical schemes, the nucleotide sequence of the upstream primer is shown as SEQ ID NO: 17, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 18, respectively.

4. A recombinant cell transformed from the recombinant expression vector into a recipient cell.

5. A foreign protein expressed by the recombinant cell.

6. The application of the promoter of the bombyx mori peyronie inducible expression gene BmPGT 3 in breeding anti-peyronie bombyx mori.

The invention has the beneficial effects that:

the BmPGT 3 promoter can drive exogenous genes to be expressed under the induction of nosema bombycis in silkworms, is not only suitable for molecular biological theory research such as gene function analysis, but also suitable for improving silkworm varieties by using genetic engineering, is particularly suitable for breeding silkworm anti-nosema varieties, such as induced expression endogenous lethal genes and induced gene editing systems, and has good application prospect.

Drawings

FIG. 1 is a diagram showing RT-PCR detection results, wherein N3-N48 respectively represent RT-PCR detection results of 3h, 6h, 12h, 24h and 48h after nosema bombycis infection, and C3-C48 are clear water control groups thereof;

FIG. 2 shows the transcriptional expression detection of BmPGT 3 in each tissue of Bombyx mori when Bombyx mori is not infected with Bombyx mori;

FIG. 3 shows the prediction of BmPGT 3 promoter, underlined bold bases as predicted transcription start site, underlined bold ATG as translation start site, and grey frame sequence as 5' UTR region;

FIG. 4 is a RT-PCR detection of the transcription initiation site of BmPGT 3, wherein 1-6 are the amplification results of primer pairs BmPGT 3-5' -F1/BmPGT 3-R, BmPUGT3-5' -F2/BmPGT 3-R, BmPUGT3-5' -F3/BmPGT 3-R, BmPUGT3-5' -F4/BmPGT 3-R, BmPUGT 3-F/BmPGT 3-5' -R and BmA3-F/BmA3-R, respectively;

fig. 5 is a result diagram of red fluorescence luminescence after transfection of a pSL [ PPUGT3-mCherry-SV40] expression vector by a bnn-SWU 1 cell, where a is a result diagram of fluorescence observation after addition of bombyx mori nosema induced after transfection of the expression vector, B is a result diagram of white light observation after addition of bombyx mori nosema induced after transfection of the expression vector, C is a result diagram of fluorescence observation after addition of PBS as a control after transfection of the expression vector, and D is a result diagram of white light observation after addition of PBS as a control after transfection of the expression vector.

Detailed Description

Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings. The experimental procedures, for which specific conditions are not indicated in the examples, are generally carried out according to conventional conditions, for example those described in the molecular cloning protocols (third edition, edited by J. SammBruke et al), or according to the conditions recommended by the manufacturer.

Example 1

Obtaining the gene sequence of bombyx mori pebrine induced expression gene BmUGT 3

According to the silkworm genome database SilkDB (https://silkdb.bioinfotoolkits.net/main/ species-info/-1) And kaikobase (https://kaikobase.dna.affrc.go.jp/) And obtaining a CDS sequence (SEQ ID NO:1) of the BmPGT 3(BGIBMGA010295) gene, designing detection primers BmPGT 3-F and BmPGT 3-R of the gene according to the gene sequence of BmPGT 3, using the silkworm Actin 3 gene as a reference, and using the primers BmA3-F and BmA 3-R.

BmPUGT3-F：5'-atggagattggattaaaagtag-3'(SEQ ID NO：2)

BmPUGT3-R：5'-ttacttttgattggacaaaacc-3'(SEQ ID NO：3)

BmA3-F：5'-atggtgcgctcctccaagaacg-3'(SEQ ID NO：4)

BmA3-R：5'-ctacaggaacaggtggtggcgg-3'(SEQ ID NO：5)

The normal large silkworm breed is bred in a standard environment by using an artificial feed (the temperature is 25 ℃, the humidity is 80%), 5-year-old silkworms are bred, one part of the silkworms are fed with silkworm nosema bombycis, and the other part of the silkworms are fed with clear water as a control. After feeding for 3h, 6h, 12h, 24h and 48h, respectively taking the intestines, extracting RNA by using an RNA extraction kit (R6934, OMEGA), then carrying out reverse transcription on the RNA into cDNA by using a reverse transcription kit (A5001, Promega), then using the cDNA as a template, using BmUGT 3-F and BmUGT 3-R as primers, carrying out PCR amplification by using rTaq enzyme (R004, Takara), using a BmA3-F and BmA3-R primer pair as controls, and carrying out amplification under the following conditions:

and carrying out agarose gel detection on the PCR product. As a result, as shown in FIGS. 1 and 2, BmPuGT3 detected the transcription only at 24 and 48 hours after infection, but did not detect the transcription of the gene without infection; and transcription of the gene was not detected in each tissue of normal silkworms. Thus, BmPuQT 3 can be really induced to express by the corpuscle worm.

Total RNA was extracted from the medium intestine of the silkworm infected with the above-mentioned antigen for 48 hours, and the RNA was inverted into cDNA using a reverse transcription kit (A5001, Promega) using 3' race oligo dt as a primer. The cDNA is taken as a template, BmPGT 3-F and GenRacer 3' primer are taken as upstream and downstream primers, Q5 high fidelity enzyme (M0493, NEB) is used for PCR amplification, and the amplification conditions are as follows:

3'race oligo dt：5'-gctgtcaacgatacgctacgtaacggcatgacagtgtttttttttttttttttt-3'(SEQ ID NO：6)

GenRacer 3'primer：5'-gctgtcaacgatacgctacgtaacg-3'(SEQ ID NO：7)

the PCR product was recovered with a gel recovery kit (D2500, OMEGA), and PCR was performed using the PCR product as a template and BmPGT 3-race-F2 and GenRacer 3'Nested primer as upstream and downstream primers, and the amplification conditions were as described above, and the amplified product was sequenced to obtain the sequence of BmPGT 3 gene 3' end (SEQ ID NO: 8).

BmPUGT3-race-F2：5'-tcccacgtttcttctggagc-3'(SEQ ID NO：9)

GenRacer 3'Nested primer：5'-cgctacgtaacggcatgacagtg-3'(SEQ ID NO：10)

Using website (http:// www.cbs.dtu.dk/services/Promoter /) to analyze BmPGT 3 gene sequence, found that 1931bp upstream base sequence of translation initiation codon ATG had 4 putative transcription initiation sites as shown in FIG. 3 (SEQ ID NO: 16), and based on this, designing upstream primers BmPGT 3-5' -F1, BmPGT 3-5' -F2, BmPGT 3-5' -F3, BmPGT 3-5' -F4 and BmPGT 3-F (SEQ ID NO: 2) for amplifying the 5' end sequence of the gene, all the upstream primers were paired with downstream primer BmPGT 3-R (SEQ ID NO: 3), and the pair of primers B3-F and BmA3-R was used as positive control, and PCR amplification was performed using the above cDNA as template, and the amplification conditions were as described above. As shown in FIG. 4, only the primers BmPGT 3-5'-F3, BmPGT 3-5' -F4 and BmPGT 3-F were efficiently amplified, and the amplified product was sequenced to obtain the full-length cDNA sequence of BmPGT 3 gene (SEQ ID NO: 15). In conclusion, the gene is composed of 5 exons by aligning with the sequence of the silkworm genome, the ORF of the gene has 756 bases, the gene codes 252 amino acids, the 5 'UTR of the gene has 468bp, and the 3' UTR of the gene has 298 bp.

BmPUGT3-5'-F1：5'-tccttaaactaaatataaag-3'(SEQ ID NO：11)

BmPUGT3-5'-F2：5'-atcaaatctagtatctgagctc-3'(SEQ ID NO：12)

BmPUGT3-5'-F3：5'-acgaacaaagtgtcacgttc-3'(SEQ ID NO：13)

BmPUGT3-5'-F4：5'-gaccgagattttattttaatttg-3'(SEQ ID NO：14)

EXAMPLE 2

Cloning of BmPGT 3 promoter and construction of expression vector thereof

The position of the translation initiation site ATG of the BmPGT 3 gene is determined in example 1, then the 1931bp genome sequence before the translation initiation site ATG of the BmPGT 3 gene is obtained according to the silkworm genome database, the sequence is analyzed by a website (https:// www.fruitfly.org/SEQ _ tools/promoter. html), a typical promoter structural region (SEQ ID NO: 16) is found in the sequence, and the PPUGT3 promoter specific primers PPUGT3-F-EcoR I and PPUGT3-R-BamH I of the BmPGT 3 gene are designed according to the sequence to amplify the sequence.

PPUGT3-F-EcoR I：5’-ccggaattc acctaaatcttctgtacgcc-3’(SEQ ID NO：17)

PPUGT3-R-BamH I：5’-cgcggatcccattgaagatgaccatatgaat-3’(SEQ ID NO：18)

Extracting the genome DNA of the silkworm large-sized strain by using a kit (D3396, OMEGA), and performing PCR amplification by using primers PPUGT3-F-EcoR I and PPUGT3-R-BamH I by using the silkworm large-sized genome DNA as a template, wherein the amplification conditions are as follows:

and (3) detecting amplified nucleic acid by electrophoresis, recovering a PCR product by using a PCR product recovery kit (D2500, OMEGA) to obtain a BmPGT 3 promoter fragment, connecting a target fragment with a pMD19-T vector by using T4 ligase (M0202, NEB), transforming the target fragment into DH5a competent cells, obtaining a positive clone pMD19-PPUGT3, and sequencing, wherein sequencing results show that the amplified sequence is consistent with expected results.

The method comprises the steps of utilizing a constructed pSL [ MCS-mChery-SV 40] vector (on a pSLfa1180fa vector, upstream and downstream enzyme cutting sites of an SV40 termination signal sequence are Not I and Hind III respectively, and upstream and downstream enzyme cutting sites of mChery are BamH I and Not I respectively), carrying out enzyme cutting on pMD19-PPUGT3 and pSL [ MCS-mChery-SV 40] vectors through EcoR I and BamH I, recovering a PPUGT3 promoter fragment and a skeleton of pSL [ MCS-mChery-SV 40], connecting the PPUGT3 promoter fragment and the skeleton of pSL [ MCS-mChery-SV 40] with T4 DNA ligase at 16 ℃ overnight, further transforming the fragments into DH5 alpha sensitive cells, and obtaining the positive clone of the PPUGT mediated HEmChery expression vector [ pSL [ PPUGT 3-mChery-SV 40 ].

EXAMPLE 3

Functional verification of BmPuQT 3 promoter

The pSL [ PPUGT3-mCherry-SV40] vector constructed in example 2 was transfected into the BmN-SWU1 cell line by liposome at a liposome to plasmid transfection ratio of 2: 1 ul: ug. Culturing at 28 deg.C, adding Bombyx mori at a number ratio of Bombyx mori to cell of 10:1 after 12h, adding PBS as control, and detecting red fluorescence by fluorescence microscope after 72 h. As shown in A, B, C, D in fig. 5, in cells transfected with pSL [ PPUGT3-mCherry-SV40], when bombyx mori granulomatoda was added, the promoter PPUGT3 was activated and induced to express its downstream mCherry gene, so that the cells exhibited red fluorescence, whereas the control PBS showed no red fluorescence, indicating that the promoter PPUGT3 could be induced by bombyx mori granulomatoda to activate and achieve the purpose of regulation.

Finally, the above embodiments are only intended to illustrate the technical solutions of the present invention and not to limit the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions, and all of them should be covered by the claims of the present invention.

Sequence listing

<110> university of southwest

<120> promoter of bombyx mori pebrine induced expression gene BmPGT 3 and application thereof

<160> 18

<170> SIPOSequenceListing 1.0

<210> 1

<211> 3006

<212> DNA

<213> Natural sequence (native sequence)

<400> 1

acctaaatct tctgtacgcc tatttggacc aagttttact acatattctc ttaaatattt 60

taaattagca tgtaaatctc gcaattatcg tgacatcatt tccttaaact aaatataaag 120

aaatttcgca tgaactaaat aataacaaca tatactcgta atagagaata ctaaaataac 180

ggaaaaaaaa tattacaata tcaaagcaaa aacttacatt gcagtaagca cgtatttact 240

gtcaacgtat atgtagttca tgacttctta cgtagcatgg gtgaatgagc tcctcagcat 300

tggactcgag ctgactccta gctgctctat ctcggacccg tacaatttat ctcccggcgc 360

cctgcatatc ctctcatcat aaggcttgag cattctcccg gctcccggca gcaacaccct 420

tttctgtctt cttctattgt tctcatcgct tcatccaaat caatcttctg tgaggttttt 480

tttttctttt tctgtaattt gaaaatcttc tttcctctta tttctcgtct tcacaaactt 540

ctattcttca taatattctt cataattata tttcttcata ttttattcag ctactctcgt 600

attttacgta tgttgcaaaa attttaactg ctggctaagg gtcgccatgt attggagtgt 660

tgtaggtgaa ataaaagaga actgttcaag tcgacaattc gtgtaatact attataattt 720

agtgtgtgca gttacaaata taaggttaga tgagctgcac actacaatca tttcagccta 780

tcgccgtcca ctgctggaca taggcctctc caatagattt ccagtgcgac cggtccattg 840

ccacctgcat ccaacgagac ccagcgcttt ttactaggcc gtcggtccat ccagtaggtg 900

gccgtcccac actgcgcttg ccagtacgtg gtcgccactc caggatcttt ctgccccatc 960

gaccgtcctc tcttcgtgca atgtatacgg acatatatta ttataattac ttttatattt 1020

atagatgaat cttatatttc agactccacg ttcactatag aaaagctaat gcacaaaact 1080

catgcaggaa tggaaatgtt taaacttgtc aagtctttta ttaatacagc taatgataca 1140

gtatctaata cggaagtaca acagctgatg ttagatccgc agacacattt tgatgttgtt 1200

attgcagaat ggatggtaac tgaaattttt agtgggtaag atgaaacaat aattaataag 1260

cagagaaaac ctaataaata aaacaattgc gtaatgacga tcactgctac agataattaa 1320

ttatttgaaa ccaaatgaag tcacgataaa aaaagctaaa taagaagtcg ttaaataaat 1380

caaatctagt atctgagctc aaacccatta tcatattttt tgaagtgata gcgtctaaaa 1440

atcgatgagc accagctgca tacacgaaca aagtgtcacg ttcctcctga gcctgagcgt 1500

gcaagcgaga gcgcggaaca agcaatagag aggcacgatc ggcccaagcg ttgccttgtg 1560

acatattttt ttctcatctc gcgtgacgtc agagctagca gttctgatag ttctgctact 1620

aacgttaaac gtaaaacgtc cgtaaaccaa ttttacagcg accgagattt gagcaccagc 1680

tgcatactta ttttaatttg aaactcctct ttttgatctg attataggtg aaaaaaatgt 1740

tcacaccgtt accatcgtat cagagctcaa ttaaaaaaat ttacacatta ttacggtatc 1800

acgaaattat ctgctgtggg cctcatattt ctattttaga ttataagagt tattatggat 1860

attcgtctat caatatgtaa ctggaatatg atgttttttc agcttcggta aaattttcaa 1920

ctgtcctata attcatatgg tcatcttcaa tggagattgg attaaaagta gccaaatggt 1980

tttccatttc aatagaagaa catatttata aagaaggatt tgctgctgca ttcaaagcaa 2040

aaggtctcgt tcagcctagc ttggaagaat tgagatactc tgctgctttg gttttgggaa 2100

attcccacgt ttcttctgga gctccgctga cattgccaca aaattacaag gctattggtg 2160

gttatcatat agacgaacaa tctaagccat tgcccaagga atttaagaat attttagaca 2220

actcgaagca tggcgttatt tatttcagtc taggatcggt agtttcaagt aaatcgatgc 2280

ctgcagcaat caaaaccgga ttatttgaaa tgttcaggag tttaaaatat actgttatat 2340

ggaaattcga agatgacttt caaaatattc ctgataacgt tcacgtcgta aaatgggcac 2400

cacagcaaag catactagca catcctaact gcattctctt catcacccac ggtggcttat 2460

tgtctacaac ggaaacatta cattacggtg ttcctattat tggaataccc atatttggag 2520

atcaggtcat gaatatcaaa aaggctgtcc ataaaggcat tggactagaa gtgaaacttg 2580

acttggatac tccaaagaac ttgaaagcag ctataaatga ggttttgtcc aatcaaaagt 2640

tgaaaataaa ttactataaa ataaataact gggaacaaat tgcgcacgat cattcagccg 2700

caatttagga ttccctgggt tgtgggtacc agagactgat aaaaatactt atatatgttt 2760

aaatgtatac ataaatagat aataaatacc cagaacaaca gcaaacaaac ctattctaac 2820

acagagatcg tttttgccac acacccaagg tgtttcctga gctctacgcc atttcttcaa 2880

acgaggatcg actataatca taccatggtg tcacctaaga gtgaatgcaa tcactagatc 2940

acagtcaccg agatgtctgt tagctcgtag gcatataaaa ataaataaac tattattatg 3000

taacct 3006

<210> 2

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

atggagattg gattaaaagt ag 22

<210> 3

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

ttacttttga ttggacaaaa cc 22

<210> 4

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

atggtgcgct cctccaagaa cg 22

<210> 5

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

ctacaggaac aggtggtggc gg 22

<210> 6

<211> 54

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

gctgtcaacg atacgctacg taacggcatg acagtgtttt tttttttttt tttt 54

<210> 7

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

gctgtcaacg atacgctacg taacg 25

<210> 8

<211> 1057

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

atggagattg gattaaaagt agccaaatgg ttttccattt caatagaaga acatatttat 60

aaagaaggat ttgctgctgc attcaaagca aaaggtctcg ttcagcctag cttggaagaa 120

ttgagatact ctgctgcttt ggttttggga aattcccacg tttcttctgg agctccgctg 180

acattgccac aaaattacaa ggctattggt ggttatcata tagacgaaca atctaagcca 240

ttgcccaagg aatttaagaa tattttagac aactcgaagc atggcgttat ttatttcagt 300

ctaggatcgg tagtttcaag taaatcgatg cctgcagcaa tcaaaaccgg attatttgaa 360

atgttcagga gtttaaaata tactgttata tggaaattcg aagatgactt tcaaaatatt 420

cctgataacg ttcacgtcgt aaaatgggca ccacagcaaa gcatactagc acatcctaac 480

tgcattctct tcatcaccca cggtggctta ttgtctacaa cggaaacatt acattacggt 540

gttcctatta ttggaatacc catatttgga gatcaggtca tgaatatcaa aaaggctgtc 600

cataaaggca ttggactaga agtgaaactt gacttggata ctccaaagaa cttgaaagca 660

gctataaatg aggttttgtc caatcaaaag ttgaaaataa attactataa aataaataac 720

tgggaacaaa ttgcgcacga tcattcagcc gcaatttagg attccctggg ttgtgggtac 780

cagagactga taaaaatact tatatatgtt taaatgtata cataaataga taataaatac 840

ccagaacaac agcaaacaaa cctattctaa cacagagatc gtttttgcca cacacccaag 900

gtgtttcctg agctctacgc catttcttca aacgaggatc gactataatc ataccatggt 960

gtcacctaag agtgaatgca atcactagat cacagtcacc gagatgtctg ttagctcgta 1020

ggcatataaa aataaataaa ctattattat gtaacct 1057

<210> 9

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

tcccacgttt cttctggagc 20

<210> 10

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

cgctacgtaa cggcatgaca gtg 23

<210> 11

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

tccttaaact aaatataaag 20

<210> 12

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

atcaaatcta gtatctgagc tc 22

<210> 13

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

acgaacaaag tgtcacgttc 20

<210> 14

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

gaccgagatt ttattttaat ttg 23

<210> 15

<211> 1526

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

acgaacaaag tgtcacgttc ctcctgagcc tgagcgtgca agcgagagcg cggaacaagc 60

aatagagagg cacgatcggc ccaagcgttg ccttgtgaca tatttttttc tcatctcgcg 120

tgacgtcaga gctagcagtt ctgatagttc tgctactaac gttaaacgta aaacgtccgt 180

aaaccaattt tacagcgacc gagattttat tttaatttga aactcctctt tttgatctga 240

ttataggtga aaaaaatgtt cacaccgtta ccatcgtatc agagctcaat taaaaaaatt 300

tacacattat tacggtatca cgaaattatc tgctgtgggc ctcatatttc tattttagat 360

tataagagtt attatggata ttcgtctatc aatatgtaac tggaatatga tgttttttca 420

gcttcggtaa aattttcaac tgtcctataa ttcatatggt catcttcaaa tggagattgg 480

attaaaagta gccaaatggt tttccatttc aatagaagaa catatttata aagaaggatt 540

tgctgctgca ttcaaagcaa aaggtctcgt tcagcctagc ttggaagaat tgagatactc 600

tgctgctttg gttttgggaa attcccacgt ttcttctgga gctccgctga cattgccaca 660

aaattacaag gctattggtg gttatcatat agacgaacaa tctaagccat tgcccaagga 720

atttaagaat attttagaca actcgaagca tggcgttatt tatttcagtc taggatcggt 780

agtttcaagt aaatcgatgc ctgcagcaat caaaaccgga ttatttgaaa tgttcaggag 840

tttaaaatat actgttatat ggaaattcga agatgacttt caaaatattc ctgataacgt 900

tcacgtcgta aaatgggcac cacagcaaag catactagca catcctaact gcattctctt 960

catcacccac ggtggcttat tgtctacaac ggaaacatta cattacggtg ttcctattat 1020

tggaataccc atatttggag atcaggtcat gaatatcaaa aaggctgtcc ataaaggcat 1080

tggactagaa gtgaaacttg acttggatac tccaaagaac ttgaaagcag ctataaatga 1140

ggttttgtcc aatcaaaagt tgaaaataaa ttactataaa ataaataact gggaacaaat 1200

tgcgcacgat cattcagccg caatttagga ttccctgggt tgtgggtacc agagactgat 1260

aaaaatactt atatatgttt aaatgtatac ataaatagat aataaatacc cagaacaaca 1320

gcaaacaaac ctattctaac acagagatcg tttttgccac acacccaagg tgtttcctga 1380

gctctacgcc atttcttcaa acgaggatcg actataatca taccatggtg tcacctaaga 1440

gtgaatgcaa tcactagatc acagtcaccg agatgtctgt tagctcgtag gcatataaaa 1500

ataaataaac tattattatg taacct 1526

<210> 16

<211> 1934

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16