CN112608919B - Promoter of bombyx mori pebrine induced expression gene BmPuGT3 and application thereof - Google Patents
Promoter of bombyx mori pebrine induced expression gene BmPuGT3 and application thereof Download PDFInfo
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Abstract
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.
Description
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. On one hand, the expression efficiency of the exogenous gene is reduced by using the constitutive promoter, and on the other hand, the constitutive promoter brings burden to the silkworm. At present, there are few reports about the Bombyx mori inducible promoter. Therefore, the development of the bombyx mori micro-particle insect 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 micro-particle insect resistance system.
Disclosure of Invention
In view of the above, an object of the present invention is to provide a promoter of bombyx mori peyronie inducible expression gene bmpgt 3, a second object of the present invention is to provide a recombinant expression vector, a third object of the present invention is to provide a method for constructing the recombinant expression vector, a fourth object of the present invention is to provide a recombinant cell, a fifth object of the present invention is to provide a foreign protein, and a sixth object of the present invention is to provide an application of the promoter of bombyx mori peyronie inducible expression gene bmpgt 3 in breeding of anti-peyronie 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. Designing upstream and downstream primers containing enzyme cutting sites of an 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 in SEQ ID NO: 17, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 18, respectively.
4. A recombinant cell obtained by transforming a recipient cell with the recombinant expression vector.
5. An exogenous protein expressed by said recombinant cell.
6. The application of the promoter of the bombyx mori peyronie inducible expression gene BmPuGT3 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 of RT-PCR detection results, N3-N48 respectively represent RT-PCR detection results of 3h, 6h, 12h, 24h and 48h after the nosema bombycis is infected with silkworm, and C3-C48 are clear water control groups of the nosema bombycis;
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 shows the result of amplification of BmPuGT3-5' -F1/BmPuGT3-R, BmPUGT3-5' -F2/BmPuGT3-R, BmPUGT3-5' -F3/BmPuGT3-R, BmPUGT3-5' -F4/BmPuGT3-R, BmPUGT3-F/BmPuGT3-5' -R and BmA3-F/BmA3-R primer pairs, respectively, 1-6 by RT-PCR to detect the transcription initiation site of BmPuGT 3;
fig. 5 is a result diagram of red fluorescence luminescence after transfection of pSL [ PPUGT3-mCherry-SV40] expression vectors with BmN-SWU1 cells, a is a result diagram of fluorescence observation after addition of bombyx mori nosema induced after transfection of expression vectors, B is a result diagram of white light observation after addition of bombyx mori nosema induced by transfection of expression vectors, C is a result diagram of fluorescence observation after addition of PBS as a control after transfection of expression vectors, and D is a result diagram of white light observation after addition of PBS as a control for transfection of expression vectors.
Detailed Description
Hereinafter, preferred embodiments of the present invention will be described in detail 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/) The CDS sequence (SEQ ID NO:1) of BmPuGT3(BGIBMGA010295) gene is obtained, detection primers BmPuGT3-F and BmPuGT3-R of the gene are designed according to the gene sequence of BmPuGT3, the silkworm Actin 3 gene is an internal reference, and the primers are 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)
Feeding normal silkworm large breed variety with artificial feed in standard environment (temperature: 25 deg.C, humidity: 80%), starting silkworm at 5 th instar, feeding one part with nosema bombycis, and feeding the other part with clear water as control. Taking out the intestines after feeding for 3h, 6h, 12h, 24h and 48h respectively, extracting RNA by using an RNA extraction kit (R6934, OMEGA), then reversely transcribing the RNA into cDNA by using a reverse transcription kit (A5001, Promega), further taking the cDNA as a template, taking BmPuGT3-F and BmPuGT3-R as primers, carrying out PCR amplification by using rTaq enzyme (R004, Takara), taking BmA3-F and BmA3-R primer pair as controls, and carrying out amplification conditions as follows:
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, BmPuGT3 is proved to be induced to express by the corpuscle insect.
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 BmPuQT 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 BmPuQT 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 there are 4 putative transcription initiation sites in 1931bp upstream of translation initiation codon ATG 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 BmPGT 3-F and B3-R primer pair were positive controls, 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 BmPuGT3 promoter and construction of expression vector thereof
The position of the translation initiation site ATG of BmPGT 3 gene is determined in example 1, then according to the silkworm genome database, the genome sequence 1931bp before the translation initiation site ATG of BmPGT 3 gene is obtained, 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 sequence is amplified by PPUGT3 promoter specific primers PPUGT3-F-EcoR I and PPUGT3-R-BamH I of the BmPGT 3 gene.
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-scale 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-scale 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 the 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-mCherry-SV40] 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 mCherry are BamHI and Not I respectively), carrying out enzyme cutting on pMD19-PPUGT3 and pSL [ MCS-mCherry-SV40] vectors through EcoR I and BamH I, recovering a PPUGT3 promoter fragment and a skeleton of pSL [ MCS-mCherry-SV40], connecting the PPUGT3 promoter fragment and the pSL [ MCS-mCherry-SV40] skeleton with T4 DNA ligase at 16 ℃ overnight, and further transforming the fragments into DH5 alpha competent cells to obtain the PPUGT mediated positive clone of the herCister expression vector [ pSL [ PPUGT 3-mChery-SV 40 ].
EXAMPLE 3
Functional verification of BmPuQT 3 promoter
The pSL [ PPUGT 3-mChery-SV 40] 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 the cells transfected with pSL [ PPUGT3-mCherry-SV40], when bombyx mori corpuscle was added, the promoter PPUGT3 was activated and induced to express the downstream mCherry gene, so that the cells showed red fluorescence, while the control group PBS showed no red fluorescence, which indicates that the promoter PPUGT3 was inducible to be activated by bombyx mori corpuscle, thereby achieving 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
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<120> bombyx mori corpuscle induced expression gene BmPuGT3 promoter and application thereof
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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
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 tattttaatt 1680
tgaaactcct ctttttgatc tgattatagg tgaaaaaaat gttcacaccg ttaccatcgt 1740
atcagagctc aattaaaaaa atttacacat tattacggta tcacgaaatt atctgctgtg 1800
ggcctcatat ttctatttta gattataaga gttattatgg atattcgtct atcaatatgt 1860
aactggaata tgatgttttt tcagcttcgg taaaattttc aactgtccta taattcatat 1920
ggtcatcttc aatg 1934
<210> 17
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
ccggaattca cctaaatctt ctgtacgcc 29
<210> 18
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
cgcggatccc attgaagatg accatatgaa t 31
Claims (7)
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 upstream primer has a nucleotide sequence as set forth in SEQ ID NO: 17, and 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. The use of the promoter of claim 1 for breeding bombyx mori resistant to microparticulate insects.
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