CN101016518B - Biological insecticide screening model and its preparing method and application - Google Patents
Biological insecticide screening model and its preparing method and application Download PDFInfo
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- CN101016518B CN101016518B CN2006101220574A CN200610122057A CN101016518B CN 101016518 B CN101016518 B CN 101016518B CN 2006101220574 A CN2006101220574 A CN 2006101220574A CN 200610122057 A CN200610122057 A CN 200610122057A CN 101016518 B CN101016518 B CN 101016518B
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Abstract
The invention discloses a biological insecticide screening mode, which comprises the following steps: integrating acceptor EcR with ecdysone GAP with gene expressing box Promoter-EcR-AOX1 TT and super spiral protein USP gene with gene expressing box TEF1 Promoter-USP-CYC1 TT on the colorant layer of Pasteur pichia; integrating DNA fragment with fruit fly 5XEcRE-HSP27 Promoter-GFP on the colorant layer yeast; getting the product. To co-culture screening drug with this product, we can get stopping or reinforcing GFP expression biological insecticide.
Description
Invention field
The present invention relates to pesticide field, be specifically related to utilize gene engineering method that biotic pesticide are carried out extensive method for screening.
Background technology
Along with the influence of global physical environment variation and mankind's activity, the propagation of vector borne diseases has the trend that increases the weight of.The method that the control of sanitary insect pest at present mainly uses chemistry to kill though chemical insecticide is bigger to sanitary insect pest and hydrobiont toxicity, Mammals is also had injury, and sanitary insect pest develops immunity to drugs to chemical insecticide easily.
Insect growth regulator(IGR) is nearly biotic pesticide that grew up in 20 years, and to environment and person poultry safety, prevention effect is good, can also prevent that insect from developing immunity to drugs, and can reach the purpose of long-term control, thereby application prospect is very wide.But screening of insect growth regulator is that object screens with whole polypide usually at present, and a little less than this screening method screening signal, sensitivity is low, and action target spot and mechanism are not clear, have influenced screening efficiency greatly.Therefore, need a kind of effective ways that can screen on a large scale of research.
Summary of the invention
Because growing of insect is realizations of casting off a skin by regular, the process of casting off a skin be at sterol, (20-hydroxyecdysone 20E) etc. finishes under the adjusting of several insect hormones to protect children's element, 20-HE.The acceptor of 20E is a kind of nucleic acid acceptor, by ecdysone receptor (ecdysteriod receptor, EcR) and Mammals retinoid X receptor analogs superhelix albumen (Ultraspiracleprotein USP) forms, the former is leading DNA binding site, and the latter is the ligand-binding site point.Only at EcR and USP when forming dimer, but the EcR-USP dimer could (Ecdysone responseelement EcRE) combines and starts transcribing of early gene with the moulting hormone response element that is positioned at moulting hormone induced gene promoter district.Any interference to the biochemical metabolism step of this hormonal dependent all can cause target insect growth heteroplasia even death.
Therefore, the purpose of this invention is to provide a kind of biological insecticide screening model, this model have the screening signal strong, highly sensitive, action target spot and mechanism are clear and definite, the advantage that screening efficiency is high.
The present invention realizes that the technical scheme of above-mentioned purpose is:
A kind of biological insecticide screening model, this model are the pichia pastoris phaffs that is integrated with following exogenous gene sequence on the karyomit(e):
(1) nucleotide sequence such as the described gene fragment of SEQ IDNO.1;
(2) nucleotide sequence such as the described gene fragment of SEQ IDNO.2;
(3) nucleotides sequence is classified the described gene fragment of SEQ IDNO.3 as.
A kind of biological insecticide screening model of the present invention, wherein SEQ ID NO.1 be can be in pichia pastoris phaff the expression casette GAP Promoter-EcR-AOX1TT of constitutive expression Aedes albopictus (A.albopictus) ecdysone receptor EcR, its base sequence is:
agatcttttt?tgtagaaatg?tcttggtgtc?ctcgtccaat?caggtagcca?tctctgaaat 60
atctggctcc?gttgcaactc?cgaacgacct?gctggcaacg?taaaattctc?cggggtaaaa 120
cttaaatgtg?gagtaatgga?accagaaacg?tctcttccct?tctctctcct?tccaccgccc 180
gttaccgtcc?ctaggaaatt?ttactctgct?ggagagcttc?ttctacggcc?cccttgcagc 240
aatgctcttc?ccagcattac?gttgcgggta?aaacggaggt?cgtgtacccg?acctagcagc 300
ccagggatgg?aaaagtcccg?gccgtcgctg?gcaataatag?cgggcggacg?catgtcatga 360
gattattgga?aaccaccaga?atcgaatata?aaaggcgaac?acctttccca?attttggttt 420
ctcctgaccc?aaagacttta?aatttaattt?atttgtccct?atttcaatca?attgaacaac 480
tatgaattca?tgatgaaaag?aagatggtcc?aacaatggtg?gattcactgc?tttgagaatg 540
ctggacgact?cttcttccga?ggttacttcc?tcttccgccg?ccctgggtat?gaccatgtct 600
ccaaattctc?tcggttcacc?aaactacgat?gaactggaac?tgtggagttc?ttacgaagac 660
aatgcctaca?acggtcactc?tgtcctatct?aatggtaaca?acaacctggg?aggttgtgga 720
gctgccaaca?acctgctgat?gaacggtatc?gtcggtaaca?acaacttgaa?cggaatgatg 780
aacatggcct?ctcaagctgt?ccaggccaac?gcaaactcta?ttcagcacat?cgtcggtaac 840
ctgatcaacg?gtgtgaaccc?aaatcagacc?ttgattccac?ctctgccttc?tattatccag 900
aataccttga?tgaacacccc?aagatctgaa?tccgtcaact?ccatttcttc?aggtagagag 960
gatttgtctc?cttcctcttc?attgaatggt?tacaccgacg?gttctgacgc?caagaagcag 1020
aagaagggac?caactccaag?acagcaggag?gaactgtgtc?tggtttgtgg?tgatagagct 1080
tccggttatc?actacaatgc?cttgacctgt?gagggttgta?aaggtttctt?tagaagatct 1140
gtcaccaaga?atgctgttta?ctgttgtaag?ttcggtcacg?cctgtgagat?ggacatgtac 1200
atgagaagaa?agtgtcaaga?gtgtagattg?aagaagtgtc?tggccgtcgg?aatgagacct 1260
gagtgtgtcg?tgccagagaa?ccagtgtgcc?atcaagagaa?aggagaagaa?agcccagaag 1320
gagaaggaca?aggtgcaaac?taacgccacc?gtctctacaa?ctaactctac?ctacagatct 1380
gagatactgc?caatcctgat?gaaatgtgat?ccaccgccgc?accaagctat?acctctacta 1440
ccagaaaagt?tgctgcagga?gaatagattg?agaaacatac?ctctgctgac?tgctaaccaa 1500
atggccgtca?tctacaaatt?gatctggtac?caggacggtt?acgagcagcc?atctgaggaa 1560
gatttgaaaa?gaataatgat?cggttctcca?aacgaggagg?aagatcaaca?tgacgtgcac 1620
ttcagacaca?taactgaaat?cacaatcttg?acagttcaat?tgatcgtgga?gttcgccaag 1680
ggactgccag?catttaccaa?gattccacag?gaggaccaga?tcactctgct?gaaggcctgt 1740
tcttctgagg?tcatgatgct?gagaatggcc?agaagatacg?acgctgccac?cgactccatc 1800
ctgttcgcta?acaacagatc?ttacactaga?gactcctaca?gaatggccgg?catggctgac 1860
actatagagg?acctgctgca?cttctgtaga?cagatgttct?ccttgactgt?ggacaacgtc 1920
gagtacgcct?tgctgactgc?tatcgtcatc?ttctctgaca?gaccaggtct?ggagcaggcc 1980
gagctggtcg?agcacatcca?gtcttactac?atcgacactc?tgagaatcta?catcctgaat 2040
agacacgctg?gtgacccaaa?gtgttctgtg?atattcgcca?aactgctgtc?tatcctgacc 2100
gaactgagaa?ctttgggtaa?ccagaactct?gagatgtgtt?tctctttgaa?gctgaagaac 2160
agaaaactgc?caagattcct?ggaggagatc?tgggacgtcc?aggacatacc?tccatctatg 2220
caggcccaga?tgcactctca?tggtactcca?cagtcctctt?cctcctcttc?ctcttcctct 2280
tcctcttcct?cttccaacgg?ttcttccaat?ggtaattcta?attccaacgg?acctcatcca 2340
catcctcacg?gacagcaatt?aactccaaat?cagcagcagc?ctccacaccc?acagcagcag 2400
cagcactctc?agttacagca?agttcacgcc?aacggttctg?gatctggtgg?tgcctcttcc 2460
aacaattctt?ctagttccgg?aggtttgggt?gtccctgttg?gtttgggtgg?aggtgccttg 2520
gaccacgttt?aatctagaga?acaaaaactc?atctcagaag?aggatctgaa?tagcgccgtc 2580
gaccatcatc?atcatcatca?ttgagtttta?gccttagaca?tgactgttcc?tcagttcaag 2640
ttgggcactt?acgagaagac?cggtcttgct?agattctaat?caagaggatg?tcagaatgcc 2700
atttgcctga?gagatgcagg?cttcattttt?gatacttttt?tatttgtaac?ctatatagta 2760
taggattttt?tttgtcattt?tgtttcttct?cgtacgagct?tgctcctgat?cagcctatct 2820
cgcagctgat?gaatatcttg?tggtaggggt?ttgggaaaat?cattcgagtt?tgatgttttt 2880
cttggtattt?cccactcctc?ttcagagtac?agaagattaa?gtgagacctt?cgtttgtgcg 2940
atccg 2945
A kind of biological insecticide screening model of the present invention, wherein SEQ IDNO.2 be can be in pichia pastoris phaff the expression casette TEF1Promoter-USP-CYC1TT of constitutive expression superhelix albumen USP, its base sequence is:
cccacacacc?atagcttcaa?aatgtttcta?ctcctttttt?actcttccag?attttctcgg 60
actccgcgca?tcgccgtacc?acttcaaaac?acccaagcac?agcatactaa?attttccctc 120
tttcttcctc?tagggtgtcg?ttaattaccc?gtactaaagg?tttggaaaag?aaaaaagaga 180
ccgcctcgtt?tctttttctt?cgtcgaaaaa?ggcaataaaa?atttttatca?cgtttctttt 240
tcttgaaatt?ttttttttta?gtttttttct?ctttcagtga?cctccattga?tatttaagtt 300
aataaacggt?cttcaatttc?tcaagtttca?gtttcatttt?tcttgttcta?ttacaacttt 360
ttttacttct?tgttcattag?aaagaaagca?tagcaatcta?atctaagggc?ggaattatcg 420
gatcccgaat?tatcggatcc?catgctgaag?aaggaaaaac?caatgctgtc?tgttgctgct 480
atcatccagg?ctcagggaag?atgggataga?acattgcctc?tggcaggatt?ggcaggtttc 540
gatgccgcct?tggttggtca?catgggtcca?ctgtctccac?aggacatgaa?acctgatttg 600
aaaccagaca?tatctttgct?gaatggttct?gtgggtccat?tttctcttag?acacaactgt 660
ggtccagcct?ctccaggtgc?tttcaaccag?caggtcgctg?cagcccctca?acagcaacag 720
cagaatgtca?acaactcttt?gaactctcag?cagcaaaatt?ccggtggagg?tggaggtgga 780
ggtacaccaa?ctaccccaac?caacatgtct?caacagtacc?caccaaatca?tccattgtct 840
ggttccaagc?atctgtgttc?tatctgtggt?gatagagcct?ctggaaagca?ttacggtgtt 900
tattcttgtg?aaggatgtaa?aggatttttc?aagagaactg?ttagaaaaga?cttgtcctat 960
gcctgtagag?aggacaagaa?ctgtactata?gacaaaagac?aaagaaacag?atgtctgtac 1020
tgtagatacc?agaaatgttt?ggcctgtggt?atgaagagag?aagccgtcca?ggaggagaga 1080
caaagatctt?ccaagttttc?cattaagtct?gaagagatca?actctacctc?ttccgtgaga 1140
gacgtgacca?tcgagagaat?cactgctgct?gaacagttgt?ctgaacagaa?atctggtgat 1200
aatgctatcc?catacttgag?agtcggatcc?aactctatga?ttcctccaga?atataaggga 1260
gccgtttctc?acctttgtca?gatggtgaac?aaacaaatct?accagctgat?agactttgcc 1320
agaagattgc?cacactttac?caacttgcat?agagacgacc?aggtcatgct?gctgagatgt 1380
ggttggaacg?agatgttgat?cgctgccgtc?gcctggagat?ctatggagta?catcgaaact 1440
gaaagatccc?cagatggttc?cagaatatct?attagacagc?cacaactgat?gtgtttagga 1500
ccaaatttta?ccctgcacag?aaactctgcc?cagcaggctg?gagttgacac?tctgttcgat 1560
agaatcctgt?gtgagttggg?aatcaaaatg?aaaagactgg?atgtgaccag?agctgagctt 1620
ggagttctca?aagctatcat?actgttcaat?ccagacatta?gaggacttaa?gtgtcagaat 1680
ggagacgacg?gaatgagaga?gaaaatctac?gcctgtttgg?acgaacattg?taaacaacag 1740
catccatctg?aggatggtag?attcgctcag?cttttgctga?gattaccagc?cctgagatct 1800
atctctctca?agtgtctcga?tcacctgaac?tttataagat?tgctctctga?taaacatttg 1860
gacaatttta?tcatcgagat?gctcgatatg?ccaatgtaat?ctagacacgt?ccgacggcgg 1920
cccacgggtc?ccaggcctcg?gagatccgtc?ccccttttcc?tttgtcgata?tcatgtaatt 1980
agttatgtca?cgcttacatt?cacgccctcc?ccccacatcc?gctctaaccg?aaaaggaagg 2040
agttagacaa?cctgaagtct?aggtccctat?ttattttttt?atagttatgt?tagtattaag 2100
aacgttattt?atatttcaaa?tttttctttt?ttttctgtac?agacgcgtgt?acgcatgtaa 2160
cattatactg?aaaaccttgc?ttgagaaggt?tttgggacgc?tcgaaggctt?taatttgcaa 2220
gct 2223
A kind of biological insecticide screening model of the present invention, wherein SEQ IDNO.3 is 5 * EcRE-HSP27Promoter-GFP dna fragmentation, by 5 tumor-necrosis factor glycoproteinss of fruit bat ecdysone receptor response original paper (5 * EcRE), fruit bat heat shock protein(HSP) (HSP27) gene promoter sequence, green fluorescent protein (GFP) gene order be composed in series by said sequence, its base sequence is:
cgacaagggt?tcaatgcact?tgtcgacaag?ggttcaatgc?acttgtcgac?aagggttcaa 60
tgcacttgtc?accggtcaat?gaaaatacaa?gctctgttgc?actctgaaaa?gacagctttt 120
aaaagcgcga?taagagaaga?aaatgtttta?aataaataca?tacatacatg?tacatatgta 180
tgtactgcat?cttaactgtt?cgttttgctt?tttattcgca?aagagaaact?cccagaaaag 240
aaatgtcaag?aagtttctgg?ttctttctcc?ctctctctat?gaaaagccgg?ctgtgctaga 300
aagagccagt?agatgcgaga?gaaaactgtt?tgttgaatta?cggggcgtat?tcaaaggggc 360
ttttaaatgt?cgcttaaatt?ttaagtttga?caggctaata?attgcttgcc?tatatctaaa 420
tattattata?tttgcattag?gggatcatag?ggaaaacctt?ctctgcaggc?aaaatctaac 480
gaagatggca?accccccatc?attttattaa?agttccgtcc?ctggttgcca?tgcactagtg 540
tgtgtgagcc?cagcgtcagt?ataaaagccg?gcgtcaacgt?cgcccgagca?cagtctaaac 600
tgaaaaattg?aaggcaaacg?ttgaagcaaa?cttcgctaaa?aaaattcgaa?aaagcaaaaa 660
aaattccttt?gtctagacag?ggttgtgaat?aaagagaaaa?aaaatcaaaa?ccatggggat 720
ccaccggtcg?ccaccatggt?gagcaagggc?gaggagctgt?tcaccggggt?ggtgcccatc 780
ctggtcgagc?tggacggcga?cgtaaacggc?cacaagttca?gcgtgtccgg?cgagggcgag 840
ggcgatgcca?cctacggcaa?gctgaccctg?aagttcatct?gcaccaccgg?caagctgccc 900
gtgccctggc?ccaccctcgt?gaccaccctg?acctacggcg?tgcagtgctt?cagccgctac 960
cccgaccaca?tgaagcagca?cgacttcttc?aagtccgcca?tgcccgaagg?ctacgtccag 1020
gagcgcacca?tcttcttcaa?ggacgacggc?aactacaaga?cccgcgccga?ggtgaagttc 1080
gagggcgaca?ccctggtgaa?ccgcatcgag?ctgaagggca?tcgacttcaa?ggaggacggc 1140
aacatcctgg?ggcacaagct?ggagtacaac?tacaacagcc?acaacgtcta?tatcatggcc 1200
gacaagcaga?agaacggcat?caaggtgaac?ttcaagatcc?gccacaacat?cgaggacggc 1260
agcgtgcagc?tcgccgacca?ctaccagcag?aacaccccca?tcggcgacgg?ccccgtgctg 1340
ctgcccgaca?accactacct?gagcaccca 1369
The mechanism of action of a kind of biological insecticide screening model of the present invention is: express EcR and USP in improved pichia pastoris phaff, form the EcR-USP dimer, act on EcRE, behind the startup HSP27promoter GFP is expressed, make pichia pastoris phaff present green fluorescence; The medicine that biological insecticide screening model of the present invention filters out can influence the dimeric formation of expression, EcR-USP and EcR-USP dimer and the EcRE bonded approach of EcR or USP, cause GFP to express changing (show as fluorescence intensity that fluorescigenic yeast sends change or make the increase and decrease of fluorescence yeast number purpose), illustrate promptly that this medicine can change that the EcR-USP dimer forms and with EcRE bonded efficient, thereby influencing the expression of institute's controlling gene, is a kind of effective biotic pesticide.
The preparation method of a kind of biological insecticide screening model of the present invention, its construction strategy is seen Fig. 1, is made up of following steps:
(1) encoding sequence of synthetic Aedes albopictus EcR and USP is inserted into the pGAPZ plasmid with the double expression boxes form, obtains the pGAPZ recombinant plasmid vector, and its structure is shown in Fig. 1 (A);
(2) EcR expression cassette on the pGAPZ recombinant plasmid vector and USP expression cassette are incorporated on the karyomit(e) of pichia pastoris phaff by homologous recombination, obtain yeast A;
(3) with 5 tumor-necrosis factor glycoproteinss of fruit bat ecdysone receptor response original paper, connect the HSP27 promotor and the GFP gene that are subjected to its regulation and control successively, construct 5 * EcRE-HSP27 Promoter-GFP dna fragmentation, then this dna fragmentation is inserted the pPIC3.5K plasmid, obtain the pPIC3.5K recombinant plasmid vector, its structure is shown in Fig. 1 (B);
(4) 5 * EcRE-HSP27 Promoter-GFP dna fragmentation on the pPIC3.5K recombinant plasmid vector is incorporated on the karyomit(e) of yeast A by homologous recombination, obtains yeast B, biological insecticide screening model promptly of the present invention.
Biological insecticide screening model of the present invention is used to screen the method for Biocidal medicine, and concrete steps are as follows:
(1) in 28～30 ℃, it is 0D that 250rpm is cultured to bacterial concentration to biological insecticide screening model of the present invention in the YPD substratum
600=0.1～0.2, average mark is loaded in the test tube, add by 100 μ l medicines/ml bacterium liquid and treat screening of medicaments, establish simultaneously and add equivalent to dissolve the bacterium liquid of the solvent of this medicine be control group, placing and being cultured to bacterial concentration under 28 ℃～30 ℃, the condition of 250～260rpm is OD
600=0.8～1;
(2) draw the resulting bacterium liquid of step (1) and observe, select the medicine that the GFP expression amount is changed and get final product.
Whether the method that adopts the present invention to screen the Biocidal medicine obtains medicine is that biological insecticidal materials has following two kinds of determination methods:
1, draws bacterium liquid fluorescence microscope, can make the fluorescence intensity change of fluorescigenic pichia pastoris phaff or make the medicine of fluorescigenic pichia pastoris phaff number increase and decrease be target screening thing, otherwise be non-target screening thing;
2, detecting the GFP genetic transcription efficient apply medicine group and control group saccharomyces model by semi-quantitative RT-PCR judges and treats whether screening of medicaments is target screening thing, concrete grammar is: the total RNA that extracts the pichia pastoris phaff of step (2) gained, the synthetic cDNA of reverse transcription is as the template of amplification again, design the primer that to stablize the gene fragment of transcribing on the primer of the GFP gene that can increase and the pichia pastoris phaff karyomit(e) that can increase then, house-keeping gene Actin-1 as pichia pastoris phaff, carry out sxemiquantitative RT-PCR, amplified production comprises on GFP gene and the pichia pastoris phaff karyomit(e) can stablize the gene fragment of transcribing, last is confidential reference items can stablize the gene fragment of transcribing on the pichia pastoris phaff karyomit(e), check GFP gene transcription efficient, scan the gray scale of each band by the gel imaging scanning system, obtain the ratio of GFP gene fragment and confidential reference items gray scale, have significant change before and after this ratio dispenser, then this medicine is target screening thing, otherwise is non-target screening thing.
Because biological insecticide screening model of the present invention can filter out the medicine of the dimeric formation of expression, EcR-USP, EcR-USP dimer and the EcRE bonded approach that influence EcR or USP, it is influenced dead even that these medicines can cause insect to grow, therefore biological insecticide screening model of the present invention can be used for quick, the extensive screening of Biocidal medicine, the present invention also have the screening signal strong, the advantage highly sensitive, that the mechanism of action is clear and definite.
Description of drawings
Fig. 1 is the construction strategy figure of a kind of biological insecticide screening model of the present invention, and wherein (A) figure is the structural representation of pGAPZ recombinant plasmid vector, and (B) figure is the structural representation of pPIC3.5K recombinant plasmid vector;
Fig. 2 is the routine empty control group saccharomyces model microscopically bright field of screening (10 * 20) figure;
Fig. 3 is the routine empty control group saccharomyces model fluorescent microscope visual field of screening (10 * 20) figure;
Fig. 4 is saccharomyces model microscopically bright field (10 * 20) figure of dispenser group in the screening example;
Fig. 5 is the saccharomyces model fluorescent microscope visual field (10 * 20) figure of dispenser group in the screening example;
Fig. 6 is the sxemiquantitative RT-PCR result of dispenser group model yeast and blank group model zymic in the screening example: Lane 1 is the amplification of dispenser group, and Lane 2 is blank amplifications of organizing, and M is a 100bp DNA mark.
Embodiment
Preparation example
One, material:
1, EcR sequence (Genbank ID:AF210733), USP sequence (Genbank ID:AF210734) are changed the codon of pichia spp preference, 5 tumor-necrosis factor glycoproteinss of the response element of fruit bat ecdysone receptor, 5 * EcRE (adopts the EcRE sequence of fruit bat HSP27 promoter region, Genbank ID:Y07758), synthetic by JaRa biotechnology (Shanghai) Co., Ltd.;
2, primer: use the DNAMAN software design, the hundred victory companies that match synthesize by Beijing.
Primer 1:5 ｀-gagctccccacacaccatag-3 ｀
Primer 2: 5 ｀-gaattccgcccttagattag-3 ｀
Primer 3:5 ｀-tctagacacgtccgacggc-3 ｀
Primer 4:5 ｀-aagcttagcttgcaaattaaag-3 ｀
3, pGAPZ plasmid: available from Invitrongen company
4, pPIC3.5K plasmid: available from Invitrongen company
5, pEGFP-N1: available from U.S. clonetech company
6, pichia spp GS115: available from Invitrongen company
7, toolenzyme HindIII, SacI, BgII, Not I are all available from Takara company
Two, method:
1, with the EcR sequence of synthetic and USP sequence clone to the pUC57 plasmid, obtain EcR-pUC57 plasmid and USP-pUC57 plasmid.
2, make up the pGAPZ recombinant plasmid vector:
(1) the EcR subclone of synthetic is gone into the downstream of pGAPZ plasmid GAP promotor, make up pGAP promoter-EcR-AOX1TT reading frame.
(2) be template with the pGAP plasmid, with primer 1 and primer 2 amplification TEF1promoter sequence; With the pGAP plasmid is template, with primer 3 and primer 4 amplification CYC1TT sequences; TEF1promoter sequence and the CYC1TT sequence upstream and downstream that is inserted into USP sequence in the USP-pUC57 plasmid respectively with amplification make up the TEF1promoter-USP-CYC1TT reading frame; Reclaim goal gene TEF promoter-USP-CYC1TT sequence with HindIII and Sac I double enzymolysis, the downstream of inserting pGAP promoter-ECR-AOX1TT reading frame makes up pGAPZ-ECR-AOX1TT-TEF promoter-USP-CYCTT plasmid.
3, make up pPIC3.5K-5X EcRE-HSP27promoter-GFP plasmid:
From drosophila gene group DNA, amplify the HSP27promoter sequence, be connected to 5 * EcRE sequence downstream, then 5 * EcRE-HSP27promoter sequence is connected into the upstream of the GFP gene of pEGFP-N1 plasmid, obtain 5 * EcRE-HSP-GFP segment with BglII and Not I double enzymolysis then, and utilize identical restriction enzyme site that this fragment subclone is gone into the pPIC3.5K plasmid.
4, make up a kind of biological insecticide screening model of the present invention:
Use AvrII enzymolysis pGAPZ-ECR-AOX1TT-TEF promoter-USP-CYCTT plasmid, make plasmid linearization, gel-purified reclaims, and electricity transforms GS115 yeast, Zeocin
+The positive high copy clone of gradient plate screening obtains recombination yeast A (operation is referring to Invitrongen company Pichia anomala expression handbook).
Use Bgl II enzyme to cut pPIC3.5K-5 * EcRE-HSP27promoter-GFP plasmid, gel-purified reclaims big segment, and electricity transforms above-mentioned recombination yeast A, histidine defect type plate screening positive colony, G418
+The high copy clone of the positive plate screening of gradient, thus high copy recombination yeast B (operation is referring to Invitrongen company Pichia anomala expression handbook) obtained.
Three, result:
From cultivating 3 days Zeocin
+The positive high copy clone of upper flat plate picking (yeast B) cultivates to the YPD nutrient solution, and 29 ℃ are shaken bacterium and are cultured to logarithmic phase, the nutrient solution coated plate, and visible globular pichia pastoris phaff cell sends green fluorescence under the fluorescent microscope, sees Fig. 4.Explanation acts on response sequence EcRE with EcR gene and USP genetic transcription, translation, combination formation EcR-USP dimer, dimer, thereby start the HSP27 promotor and start model successfully foundation in the pichia pastoris phaff body that reporter gene GFP expresses, a kind of biological insecticide screening model promptly of the present invention is successfully set up.
The screening example
(1) treats screening of medicaments
Chinese: the worm hydrazides, purchase in Jiang Sushan and reach chemical industry company limited.
Common name: tebbufenozide
Trade name: MIMIC, comfirm, Romdan, rice is full
Chemical name: the N-tertiary butyl-N-(4-ethylamino benzonitrile acyl group)-3,5-dimethyl benzene hydrazides
Toxicity:, higher animal is not had teratogenesis, carcinogenic, mutagenesis to eyes and skin nonirritant.This agent is weighed by world's grain and oil tissue (FAO) standard comprehensively, true border nontoxic level Pesticidal products.
Mechanism of action: worm hydrazides insecticides is a 20-hydroxyecdysone analogue, and its mechanism of action is still indeterminate.
(2) screening method
(bacterial concentration is OD with biological insecticide screening model of the present invention
600=0.1) is sub-packed in the test tube 5 milliliters of every pipes.Stay 5 blank pipes (do not add and treat screening of medicaments, only add the solvent that 500 μ l dissolve this medicine) as control group, other experimental group adds 500 μ l and treats screening of medicaments (the dispenser process is referring to specification sheets), cultivates altogether in the incubator of 28 ℃ and 250rpm.Treat that bacterial concentration is OD
600=0.8 o'clock, draw 10 μ l and on slide glass, use fluorescence microscope.
(3) The selection result
Experimental result is seen Fig. 3, Fig. 4, Fig. 5, Fig. 6, and as can be seen from the figure, the dispenser group obviously reduces with respect to the yeast number of its fluorescence of blank group, and fluorescence zymic fluorescence intensity also obviously weakens.As seen the worm hydrazides is effective biotic pesticide.
To detect the GFP genetic transcription efficient that applies medicine group and control group saccharomyces model by semi-quantitative RT-PCR below.
A. primer: use the DNAMAN software design, it is synthetic to match hundred victory companies by Beijing.
pActinl：5＇-TGTCACCAACTGGGACGATA-3＇
pActin2：5＇-AACCAGCGTAAATTGGAACG-3＇
pGFP1：5＇-TATACGTAATGGTGAGCAAGGGCG-3＇
pGFP2：5＇-ATCCAAGGGTTGGCTAGATCCGGTGGAT-3＇
B. template
Use Omega company yeast total RNA extraction reagent box to extract dispenser group and the blank total RNA of model yeast that organizes respectively, use random primer, the synthetic cDNA of M-MLV reversed transcriptive enzyme reverse transcription, as the template of PCR reaction.
The c.PcR product
Primer pActin1 and pActin2 amplification yeast house-keeping gene Actin-1, PCR product length 283bp; Primer pGFP1 and pGFP2 amplification EGFP gene, PCR product length is 737bp.
The d.PCR reaction
Adopting the method for GFP and Aetin-1 tube amplification, is template with reverse transcription after product cDNA, adopts 54 ℃ of renaturation temperature amplification GFP, Actin-1 gene.
E. result and analysis
The PCR product the results are shown in Figure 7 with 1% agarose gel electrophoresis analysis, with pichia pastoris phaff house-keeping gene Actin-1 as confidential reference items, check GFP gene transcription efficient.Scan the gray scale of each band by the gel imaging scanning system, ask the ratio of bandl (GFP) and band2 (confidential reference items) gray scale, lane 1 ratio is 0.614, and lane 2 ratios are 1.134.As seen after applying medicine, the GFP gene transcription level of pichia pastoris phaff obviously reduces, and has just illustrated that also the worm hydrazides is effective biotic pesticide.
[nucleotides sequence tabulation
＜110〉Nanfang Medical Univ
＜120〉a kind of biological insecticide screening system and its production and application
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＜213〉artificial sequence
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＜213〉artificial sequence
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＜223〉artificial sequence description: gene segment
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Claims (3)
1. biological insecticide screening model, this model is the pichia pastoris phaff that is integrated with following exogenous gene sequence on the karyomit(e):
(1) nucleotide sequence such as the described gene fragment of SEQ ID NO.1;
(2) nucleotide sequence such as the described gene fragment of SEQ ID NO.2;
(3) nucleotide sequence such as the described gene fragment of SEQ ID NO.3;
And prepare by following:
(a) encoding sequence of synthetic Aedes albopictus EcR and USP is inserted into the pGAPZ plasmid with the double expression boxes form, obtains the pGAPZ recombinant plasmid vector;
(b) be incorporated into the double expression boxes on the pGAPZ recombinant plasmid vector on the karyomit(e) of pichia pastoris phaff by homologous recombination;
(c) with 5 tumor-necrosis factor glycoproteinss of response element of fruit bat ecdysone receptor, connect the HSP27 promotor and the GFP gene that are subjected to its regulation and control successively, construct 5 * EcRE-HSP27 Promoter-GFP dna fragmentation, then this dna fragmentation is inserted the pPIC3.5K plasmid, obtain the pPIC3.5K recombinant plasmid vector;
(d) 5 * EcRE-HSP27 Promoter-GFP dna fragmentation on the pPIC3.5K recombinant plasmid vector is incorporated on the karyomit(e) of the pichia pastoris phaff that contains EcR expression casette and USP expression casette by homologous recombination.
2. the preparation method of the described a kind of biological insecticide screening model of claim 1, this method is made up of following steps:
(1) encoding sequence of synthetic Aedes albopictus EcR and USP is inserted into the pGAPZ plasmid with the double expression boxes form, obtains the pGAPZ recombinant plasmid vector;
(2) be incorporated into the double expression boxes on the pGAPZ recombinant plasmid vector on the karyomit(e) of pichia pastoris phaff by homologous recombination;
(3) with 5 tumor-necrosis factor glycoproteinss of response element of fruit bat ecdysone receptor, connect the HSP27 promotor and the GFP gene that are subjected to its regulation and control successively, construct 5 * EcRE-HSP27 Promoter-GFP dna fragmentation, then this dna fragmentation is inserted the pPIC3.5K plasmid, obtain the pPIC3.5K recombinant plasmid vector;
(4) 5 * EcRE-HSP27 Promoter-GFP dna fragmentation on the pPIC3.5K recombinant plasmid vector is incorporated on the karyomit(e) of the pichia pastoris phaff that contains EcR expression casette and USP expression casette by homologous recombination.
3. the described biological insecticide screening model of claim 1 method that is used to screen the Biocidal medicine, this method is made up of following steps:
(1) in 28～30 ℃, it is OD that 250rpm is cultured to bacterial concentration to described biological insecticide screening model in the YPD substratum
600=0.1～0.2, average mark is loaded in the test tube, add by 100 μ l medicines/ml bacterium liquid and treat screening of medicaments, establish simultaneously and add equivalent to dissolve the bacterium liquid of the solvent of this medicine be control group, placing and being cultured to bacterial concentration under 28 ℃～30 ℃, the condition of 250～260rpm is OD
600=0.8～1;
(2) draw the resulting bacterium liquid of step (1) and observe, select the medicine that the GFP expression amount is changed and get final product.
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| CN109439685A (en) * | 2018-12-26 | 2019-03-08 | 菏泽学院 | A kind of construction method of the plant expression vector that can express gypsymoth USP gene dsRNA and its application |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1215432A (en) * | 1996-04-05 | 1999-04-28 | 索尔克生物学研究所 | Hormone-mediated methods for modulating expression of exogenous genes in mammlian system, and products related thereto |
| CN1422334A (en) * | 2000-03-22 | 2003-06-04 | 罗姆和哈斯公司 | Ecdysone receptor-based inducible gene expression system |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1215432A (en) * | 1996-04-05 | 1999-04-28 | 索尔克生物学研究所 | Hormone-mediated methods for modulating expression of exogenous genes in mammlian system, and products related thereto |
| CN1422334A (en) * | 2000-03-22 | 2003-06-04 | 罗姆和哈斯公司 | Ecdysone receptor-based inducible gene expression system |
Non-Patent Citations (2)
| Title |
|---|
| Tran H T et al..Reconstruction of ligand-dependent transactivation ofChoristoneura fumiferana ecdysone receptor in yeast..Molecular Endocrinology15 7.2001,15(7),1140-1153. |
| Tran H T et al..Reconstruction of ligand-dependent transactivation ofChoristoneura fumiferana ecdysone receptor in yeast..Molecular Endocrinology15 7.2001,15(7),1140-1153. * |
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