CN113234746B - Method for pesticide induced protein interaction and induced gene expression - Google Patents
Method for pesticide induced protein interaction and induced gene expression Download PDFInfo
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- CN113234746B CN113234746B CN202110440791.XA CN202110440791A CN113234746B CN 113234746 B CN113234746 B CN 113234746B CN 202110440791 A CN202110440791 A CN 202110440791A CN 113234746 B CN113234746 B CN 113234746B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The application discloses a method for pesticide induced protein interaction and induced gene expression, wherein the pesticide is mandipropamid, the induced protein interaction is used for inducing three fragments of green fluorescent protein to complement, and the induced gene expression is used for inducing the expression of a green fluorescent protein coding gene; the provided ABI-CP is a topological structure variant which uses mandipropamid to control protein interaction, and breaks through the limitation of the existing method on the spatial relative position of fusion protein; the method for regulating gene expression by using mandipropamid breaks through the limitation of the existing compounds for inducing gene expression due to the characteristics of pesticide compounds. These limitations include high cost, non-availability in large amounts in the environment, non-physiological neutrality (rapid metabolism), difficult cell entry, etc.
Description
Technical Field
The invention relates to the technical field of gene expression regulation, in particular to a method for pesticide induced protein interaction and induced gene expression.
Background
Protein molecular interactions are the basis for a variety of biological processes. Detection and manipulation of protein molecular interactions is a common means of detecting and controlling biological functions such as signal transduction in an organism. For example, by inducing interaction between two protein molecules using a compound molecule, the spatial position of the fluorescent protein expressed by fusion with the two proteins is brought closer, and the presence or concentration of the compound can be detected by fluorescence resonance transfer. For another example, if two proteins are induced to interact by light, the two proteins can be respectively constructed as fusion proteins with a pair of effector proteins, and the interaction and the function of the effector proteins can be controlled by utilizing light. This approach has become an important component of optogenetic technology.
The pesticide compound has incomparable advantages to the common compound. Pesticides have been approved by environmental safety management, and have clear biological and toxicological effects on animals, plants, microorganisms, and the like. Meanwhile, the pesticide has the advantages of low cost, large scale, high efficiency and the like in application. Therefore, development of a method for interacting proteins induced by pesticides makes it possible to control biological processes such as gene expression by pesticides.
Mandipropamid is a main component of a low-toxicity efficient pesticide 'Ruifen' specially developed by Zhengda company. The research group has developed proteins capable of interacting under the induction of mandipropamid, and is applied to the interaction of plant and animal cell control effector proteins so as to control biological processes such as plant hormone abscisic acid signal transduction, and the change of subcellular localization of proteins in animal cells. The existing method uses PYR1 MANDI Interact with amino-terminally truncated HABI protein (hereinafter referred to as HAB), or PYR1 MANDI And amino-terminally truncated ABI1 (hereinafter referred to as ABI, a homologous protein to the HAB1 protein).
There are two limitations to the existing methods. The first limitation is that the amino-and carboxy-termini of the ABI proteins are spatially adjacent, on the same side of the ABI protein, and PYR1 interacts with ABI MANDI The carboxyl terminal is located on the opposite side of the ABI protein, and the space distance exceeds 67 angstroms (PDB accession number 3 NMN), the steric hindrance is large, and PYR1 MANDI And ABI1, is unfavorable for pulling up effector proteins after interaction. PYR1 MANDI The amino terminus is further from the amino terminus or the carboxy terminus of the ABI protein. The second limitation is that there are few current methods of mandipropamid-induced gene transcription activation. PYR1 has been reported to be produced in yeast cells by a yeast two-hybrid system MANDI And HAB or ABI interactions into transcriptional activation signals. However, this strategy is limited by the principle of yeast two-hybrid and is not universally applicable.
Disclosure of Invention
The invention provides a method for pesticide induced protein interaction and induced gene expression, which solves the limitation of the existing induced protein interaction compound as a gene expression inducer, and uses mandipropamid to control another spatial position variant of protein interaction, thereby breaking through the limitation of the existing method on the spatial relative position of protein.
The invention adopts the following technical scheme:
a method for interacting and inducing gene expression of pesticide-induced protein, wherein the pesticide is mandipropamid, the induced protein is complementary to three fragments of the induced green fluorescent protein, and the induced gene expression is the induced green fluorescent protein coding gene expression, and the method comprises the following steps:
step 1: construction of plasmid 2 on the basis of plasmid 1, ABI-CP amino-terminal fusion GFP10, PYR1 MANDI The carboxyl end is fused with GFP11;
step 2: constructing a plasmid 3, and controlling induction expression of GFP1-9 fragments in escherichia coli by using a T7 promoter_lacO to match with an IPTG induction system of escherichia coli BL21 (DE 3) strain;
step 3: transforming the plasmids 2 and 3 into an escherichia coli BL21 (DE 3) strain;
step 5: after 3-4 hours, the fluorescence intensities of the three fragments GFP1-9, GFP10 and GFP11 recombined into GFP were measured with the following parameters, excitation light 488nm, emission light 510nm, and OD was measured simultaneously 600 The method is used for homogenizing the culture concentration of the bacterial liquid, and the result shows that mandipropamid efficiently induces the complementation of three fragments of green fluorescent protein;
step 6: construction of plasmid 4, resolved T7 RNA polymerase and PYR1 in E.coli MANDI Respectively fusion-expressing with ABI-CP;
step 7: constructing a plasmid 5, driving GFP coding gene expression by using a T7 promoter, and detecting the activity of mandipropamid-induced T7 RNA polymerase in escherichia coli;
step 8: transforming the plasmids 4 and 5 into an escherichia coli Top10 strain;
step 9, detection of expression products of plasmids 4 and 5 induced by mandipropamid in E.coli: taking positive clone shake bacteria of escherichia coli Top10 strain background, and waiting for OD 600 Reaching 0.40-0.60, adding mandipropamid with final concentration gradient of 0.1 μm,1 μm,10 μm,100 μm,200 μm,400 μm into bacterial liquid, and using the following parameters after 3-4 hrExcitation light 488nm and emission light 510nm for detecting GFP expression effect, and simultaneously measuring OD 600 The mandipropamid is used for homogenizing the culture concentration of the bacterial liquid, and the result shows that mandipropamid induces the expression of the green fluorescent protein coding gene.
As a preferred technical scheme of the invention: the plasmid 1 contains two expression cassettes, and PYR1 is respectively expressed in the escherichia coli in a constitutive mode MANDI And ABI-CP.
As a preferred technical scheme of the invention: the plasmid 2 is constructed based on the plasmid 1 and comprises two expression cassettes, namely ABI-CP of amino-terminal fusion GFP10 fragment and PYR1 of carboxyl-terminal fusion GFP11 in escherichia coli MANDI 。
As a preferred technical scheme of the invention: the plasmid 3 was induced to express GFP1-9 fragment in E.coli by IPTG (isopropyl thiogalactoside).
As a preferred technical scheme of the invention: the plasmid 4, constructed based on plasmid 1, contains two expression cassettes, which allow the split T7 RNA polymerase and PYR1 in E.coli MANDI And ABI-CP are respectively fused and expressed.
As a preferred technical scheme of the invention: the expression frame of the plasmid 5 contains a T7 promoter to drive the target gene to express, and the activity of T7 RNA polymerase after induction of mandipropamid is detected in escherichia coli.
As a preferred technical scheme of the invention: the concentration gradient of the mandipropamid with different concentrations in the step 4 is 1 mu M,10 mu M and 100 mu M.
As a preferred technical scheme of the invention: the strain with endogenous T7 RNA polymerase cannot be used in step 8.
As a preferred technical scheme of the invention: the strain with the endogenous T7 RNA polymerase is BL21 (DE 3) strain.
As a preferred technical scheme of the invention: the background of the positive clone in the step 4 is an escherichia coli BL21 (DE 3) strain, and the background of the positive clone in the step 9 is an escherichia coli Top10 strain.
As a preferred technical scheme of the invention: the concentration gradient of the mandipropamid in the step 9 is 0.1 mu M,1 mu M,10 mu M,100 mu M,200 mu M and 400 mu M.
Advantageous effects
Compared with the prior art, the method for pesticide induced protein interaction and induced gene expression has the following technical effects:
1. the provided ABI-CP is a topological structure variant which uses mandipropamid to control protein interaction, and breaks through the limitation of the existing method on the spatial relative position of fusion protein;
2. the method for regulating gene expression by using mandipropamid breaks through the limitation of the existing compounds for inducing gene expression due to the characteristics of pesticide compounds. These limitations include high cost, non-availability in large amounts in the environment, non-physiological neutrality (rapid metabolism), difficulty in entering cells for efficacy, etc.;
3. the ABI protein is subjected to cyclic rearrangement (hereinafter referred to as ABI-CP), the position of the amino terminal of the ABI is adjusted, S234 is taken as a new amino terminal of the ABI, and the original carboxyl terminal K423 and the original amino terminal V126 are connected by a flexible peptide chain; the amino terminus of ABI is near PYR1 MANDI The carboxy terminus of the protein, which is reduced to about 24 angstroms in distance, is spatially ipsilateral to the ABI-CP protein;
4. two techniques for detecting and outputting protein interaction information are used, one is that green fluorescent protein is split into three fragments of GFP1-9, GFP10 and GFP11 are respectively fused into one protein, if the two proteins interact, GFP10 and GFP11 are pulled up, GFP1-9 is recruited, the three fragments are complemented into correctly folded fluorescent protein, and a protein interaction event can be directly output as a fluorescent protein signal; the technology is used for detecting the property of the ABI-CP induced interaction by mandipropamid, and shows that the arrangement of the ABI-CP in the fusion protein is more flexible;
5. another technique is the double-piece complementation of T7 RNA polymerase, i.e. T7 RNA polymerase is split into two pieces of T7N and T7C for expression, respectively, but does not spontaneously recombine into a functional T7 RNA polymerase, when T7N and T7C are fused to a protein respectively, T7N and T7C are pulled up to complement to a correctly folded T7 RNA polymerase if the two proteins interact; the protein interaction event is output in a mode of driving the target gene to express after the T7 RNA polymerase is recombined, and the T7 RNA polymerase has universality, so that the mandipropamid induces the gene transcription activation, and therefore, the mandipropamid has universality.
6. The technology provided by the invention can be used for manipulating the gene expression of microorganisms insensitive to mandipropamid and interacting with plants (figure 8), and the application of the pesticide compound is increased.
Drawings
FIG. 1 is a schematic diagram of the principle of three-fragment complementation of mandipropamid-induced green fluorescent protein in the present application.
FIG. 2 is a block diagram showing the expression of plasmid 2 and plasmid 3 of the present application.
FIG. 3 is a graph showing the three complementary fragments of mandipropamid-induced green fluorescent protein under blue light irradiation and with an orange filter.
FIG. 4 is a graph showing the quantitative results of the mandipropamid-induced complementation of three fragments of green fluorescent protein according to the present application.
FIG. 5 is a schematic diagram of the principle of expression of the genes encoding the mandipropamid-induced green fluorescent protein in the present application.
FIG. 6 is a block diagram showing the expression of plasmid 4 and plasmid 5 of the present application.
FIG. 7 is a graph showing the quantitative results of the induction of green fluorescent protein expression by mandipropamid of the present application.
FIG. 8 is a graph showing that mandipropamid can induce the green fluorescent protein of the E.coli reporter gene in plant leaves; wherein A is a confocal laser microscope photograph of green fluorescent protein expression after induction under low-intensity excitation light; b is a confocal laser microscope photograph of the green fluorescent protein expression after the contrast DMSO treatment under the low-intensity excitation light; shooting A and B under the same parameter condition; c is a photograph of green fluorescent protein expression after the control DMSO treatment under high-intensity excitation light; b and C are the same field of view, C shows the presence of E.coli in B, scale: 50 microns.
Detailed Description
The invention is further described below with reference to examples, which are intended to be illustrative only and not to limit the scope of the claims, as other alternatives, which can be envisaged by a person skilled in the art, are within the scope of the claims.
The sequence of plasmid 1 is shown as SEQ ID NO.1, the sequence of plasmid 2 is shown as SEQ ID NO.2, the sequence of plasmid 3 is shown as SEQ ID NO.3, the sequence of plasmid 4 is shown as SEQ ID NO.4, the sequence of plasmid 5 is shown as SEQ ID NO.5, the sequence of T7N is shown as SEQ ID NO.6, and the sequence of ABI1-CP is shown as SEQ ID NO.7, PYR1 MANDI The sequence is shown as SEQ ID NO.8, the T7C sequence is shown as SEQ ID NO.9, the GFP10 sequence is shown as SEQ ID NO.10, the GFP11 sequence is shown as SEQ ID NO.11, the GFP1-9 sequence is shown as SEQ ID NO.12, the T7 promoter_lacO sequence is shown as SEQ ID NO.13, and the T7 promoter sequence is shown as SEQ ID NO. 14; the brief molecular mechanism is that mandipropamid binds PYR1 first MANDI Resulting in PYR1 MANDI Conformational changes and binding to HAB or ABI. PYR1 MANDI Wherein ABI is also a phosphatase-inactivating mutant by point mutation technique to reduce interference to host organisms. A pair of protein molecules which need to be regulated by mandipropamid are respectively regulated by PYR1 MANDI And HAB or ABI, so that mandipropamid can induce this pair of protein interactions.
Example 1:
a method for pesticide induced protein interaction and induced gene expression comprises the following steps of:
step 1: construction of plasmid 2 on the basis of plasmid 1, ABI-CP amino-terminal fusion GFP10, PYR1 MANDI The carboxy terminus fused GFP11.
Step 2: plasmid 3 was constructed and induction of GFP1-9 fragment expression was controlled by the T7 promoter_lacO in E.coli to match the IPTG induction system of E.coli BL21 (DE 3) strain.
Step 3: plasmids 2 and 3 were transformed into E.coli BL21 (DE 3) strain.
Step 5: after 3-4 hours, the fluorescence intensity of GFP1-9, GFP10 and GFP11 fragments recombined into GFP was detected with the following parameters, excitation light 488nm, emission light 510nm, and OD was measured simultaneously 600 The mandipropamid is used for homogenizing the culture concentration of bacterial liquid, and the result shows that mandipropamid efficiently induces the complementation of three fragments of green fluorescent protein.
Example 2
Mandipropamid induces the expression of the green fluorescent protein coding gene:
the first step: construction of plasmid 4, resolved T7 RNA polymerase and PYR1 in E.coli MANDI Respectively fusion-expressing with ABI-CP;
and a second step of: constructing a plasmid 5, driving GFP coding gene expression by using a T7 promoter, and detecting the activity of mandipropamid-induced T7 RNA polymerase in escherichia coli;
and a third step of: plasmids 4 and 5 were transformed into E.coli Top10 strain. Note that strains with endogenous T7 RNA polymerase, such as BL21 (DE 3) strain, cannot be used;
fourth step: detection of expression products of plasmids 4 and 5 in E.coli were induced by mandipropamid: taking positive clone shake bacteria of escherichia coli Top10 strain background, and waiting for OD 600 Reaching 0.40-0.60, adding mandipropamid with final concentration gradient of 0.1 μm,1 μm,10 μm,100 μm,200 μm,400 μm into bacterial liquid, detecting GFP expression effect with excitation light 488nm and emission light 510nm after 3-4 hr, and simultaneously determining OD 600 The mandipropamid is used for homogenizing the culture concentration of the bacterial liquid, and the result shows that mandipropamid induces the expression of the green fluorescent protein coding gene.
The previous description of the embodiments is provided to facilitate a person of ordinary skill in the art in order to make and use the present invention. It will be apparent to those skilled in the art that various modifications can be readily made to these embodiments and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above-described embodiments, and those skilled in the art, based on the present disclosure, should make improvements and modifications without departing from the scope of the present invention.
Sequence listing
<110> university of Nantong
<120> a method for pesticide-induced protein interaction and induction of gene expression
<160> 14
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ggatgacgag cgccgcgtga cgggggcatc aataatcggt ggggaacatc gccttacgaa 4920
ttataaaggc gtcactactg ttcatagatt tgagaaggaa aatagaatct ggactgttgt 4980
tctggaatct tatgtggttg atatgccaga gggtaactcg gaagatgata cacgcatgct 5040
ggcggacacg gttgtgaaac ttaatctgca aaagctggca actgtcgccg aagccatggc 5100
aggatcaggc tcgagtggtt caggatctgg tgcaagctag tcggacttcg cgttcgcgta 5160
atagcaccac caccaccacc actgagatcc ggctgctaac aaagcccgaa aggaagctga 5220
gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt 5280
cttgaggggt tttttgctga aaggaggaac tatatccgga t 5321
<210> 2
<211> 5444
<212> DNA
<213> Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 2
tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60
cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 120
ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180
gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240
acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 300
ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 360
ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 420
acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 480
tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 540
tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat 600
gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt 660
ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg 720
agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga 780
agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg 840
tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt 900
tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg 960
cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg 1020
aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga 1080
tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc 1140
tgcagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc 1200
ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc 1260
ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg 1320
cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac 1380
gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc 1440
actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt 1500
aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac 1560
caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa 1620
aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc 1680
accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt 1740
aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg 1800
ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc 1860
agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt 1920
accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga 1980
gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct 2040
tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg 2100
cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca 2160
cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa 2220
cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt 2280
ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 2340
taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 2400
gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatatgg 2460
tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat 2520
cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct 2580
gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct 2640
gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct 2700
catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt 2760
tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg 2820
ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa 2880
tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc 2940
ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa 3000
aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta 3060
gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg 3120
tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag 3180
acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac 3240
cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca 3300
cccgtggcca ggacccaacg ctgcccgaga tctcgatccc gcgaaatctg aaaggaggaa 3360
ctatactcgg atatctttac agctagctca gtcctaggta ttatgctagc gaaattaccc 3420
ttgccttaac taataagaga gctgtctatg gacctgcctg acgaccacta cctgtccacc 3480
cagaccatcc tgtccaagga cctgaacgga tctggtggat ccggctctgg ttcgagttct 3540
gtcgctcctg agaccgtggg ctctacaagt gtggtcgcag tggtcttccc atctcacatt 3600
tttgtcgcaa attgcggcga cagccgggcc gtgctgtgca ggggaaagac cgccctgcca 3660
ctgagcgtgg accataaacc cgacagggag gatgaagcag cccgcatcga ggctgcagga 3720
ggcaaagtga tccagtggaa cggagcacgg gtgtttggcg tcctggccat gtcacgcagc 3780
attggggatc gatacctgaa accatcaatc attcccgacc ctgaagtgac tgccgtcaag 3840
agagtgaaag aagacgattg cctgatcctg gctagcgacg gcgtctggga tgtgatgacc 3900
gacgaggaag catgtgagat ggcccgaaag cggattctgc tgtggcataa gaaaaatgcc 3960
gtggctggcg atgctagtct gctggcagac gagcggagaa aggaagggaa agatcccgcc 4020
gctatgagtg cagccgaata tctgtcaaaa ctggctattc agaggggcag taaggacaac 4080
atctccgtcg tcgtcgtgga cctgaagggc ggtagtggtt cgggttcttc ggtccccctg 4140
tatgggttca ccagcatttg tgggcggaga ccagagatgg aggcagccgt cagcactatt 4200
ccccggttcc tgcagtcaag ctccggcagc atgctggacg ggaggttcga tccacagtcc 4260
gccgctcact tctttggggt ctacgatgga catggcgggt cccaggtggc caactattgc 4320
agggagcgca tgcacctggc tctggcagag gaaatcgcca aggagaaacc tatgctgtgc 4380
gacggagata catggctgga aaagtggaag aaagccctgt tcaactcttt tctgcgggtg 4440
gactccgaga tcgaatgagg taccattcaa gacccccgca ccgaaaggtc cgggggtttt 4500
ttttactatt taaatcctgc cagtgttcac attcgaaccg tctctgcttt gacaacatgc 4560
tgtgcggtgt tgtaaagtcg tggccaggag aatacgacag gcaattgcaa gaaggaggat 4620
attgatgcca tcggaactta caccggagga aaggtccgag ttgaagaaca gcattgcgga 4680
gtttcataca tatcaactcg atccggggag ttgttctagt ttgcacgccc aaaggataca 4740
cgccccaccg gaacttgtgt ggagtatcgt ccgccggttc gataaaccac aaacacacag 4800
gcatttcatt aaaagctgct ccgtggagca gaactttgaa atgagagtgg gatgcacacg 4860
ggatataata gttataagcg gcctcccggc caacacgagc accgaaagat tggacatact 4920
ggatgacgag cgccgcgtga cgggggcatc aataatcggt ggggaacatc gccttacgaa 4980
ttataaaggc gtcactactg ttcatagatt tgagaaggaa aatagaatct ggactgttgt 5040
tctggaatct tatgtggttg atatgccaga gggtaactcg gaagatgata cacgcatgct 5100
ggcggacacg gttgtgaaac ttaatctgca aaagctggca actgtcgccg aagccatggc 5160
aggatcaggc tcgagtggtt caggatctgg tgcaagcgaa aagcgagacc atatggtttt 5220
gcttgagtat gttacagcgg ctggcattac cgatgcatca tagtcggact tcgcgttcgc 5280
gtaatagcac caccaccacc accactgaga tccggctgct aacaaagccc gaaaggaagc 5340
tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg 5400
ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggat 5444
<210> 3
<211> 4132
<212> DNA
<213> Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 3
agatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa 60
ttcccctcta gaaataattt tgtttaactt taagaaggag atataccatg cgcaaaggcg 120
aagaactgtt taccggcatt gtgccgattc tggtggaact ggatggcgat gtgaacggcc 180
ataaattttt tgtgcgcggc gaaggcgaag gcgatgcgac cattggcaaa ctgagcctga 240
aatttatttg caccaccggc aaactgccgg tgccgtggcc gaccctggtg accaccctga 300
cctatggcgt gcagtgcttt agccgctatc cggatcatat gaaacgccat gattttttta 360
aaagcgcgat gccggaaggc tatgtgcagg aacgcaccat ttattttaaa gatgatggca 420
cctataaaac ccgcgcggaa gtgaaatttg aaggcgatac cctggtgaac cgcattgaac 480
tgaaaggcat tgattttaaa gaagatggca acattctggg ccataaactg gaatataact 540
ttaacagcca taaagtgtat attaccgcgg ataaacagaa caacggcatt aaagcgaact 600
ttaccattcg ccataacgtg gaagatggca gcgtgcagct ggcggatcat tatcagcaga 660
acaccccgat tggcgatggc ccggttcttc ttccttagga attcttgttc agaacgctcg 720
gtcttgcaca ccgggcgttt tttctttgtg agtccaatcc aaaacgccgc gttcagcggc 780
gttttttctg cttctgaaag gaggaactat actcggatat cccgctgagc aataactagc 840
ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaac ctcaggcatt 900
tgagaagcac acggtcacac tgcttccggt agtcaataaa ccggtaaacc agcaatagac 960
ataagcggct atttaacgac cctgccctga accgacgacc gggtcatcgt ggccggatct 1020
tgcggcccct cggcttgaac gaattgttag acattatttg ccgactacct tggtgatctc 1080
gcctttcacg tagtggacaa attcttccaa ctgatctgcg cgcgaggcca agcgatcttc 1140
ttcttgtcca agataagcct gtctagcttc aagtatgacg ggctgatact gggccggcag 1200
gcgctccatt gcccagtcgg cagcgacatc cttcggcgcg attttgccgg ttactgcgct 1260
gtaccaaatg cgggacaacg taagcactac atttcgctca tcgccagccc agtcgggcgg 1320
cgagttccat agcgttaagg tttcatttag cgcctcaaat agatcctgtt caggaaccgg 1380
atcaaagagt tcctccgccg ctggacctac caaggcaacg ctatgttctc ttgcttttgt 1440
cagcaagata gccagatcaa tgtcgatcgt ggctggctcg aagatacctg caagaatgtc 1500
attgcgctgc cattctccaa attgcagttc gcgcttagct ggataacgcc acggaatgat 1560
gtcgtcgtgc acaacaatgg tgacttctac agcgcggaga atctcgctct ctccagggga 1620
agccgaagtt tccaaaaggt cgttgatcaa agctcgccgc gttgtttcat caagccttac 1680
ggtcaccgta accagcaaat caatatcact gtgtggcttc aggccgccat ccactgcgga 1740
gccgtacaaa tgtacggcca gcaacgtcgg ttcgagatgg cgctcgatga cgccaactac 1800
ctctgatagt tgagtcgata cttcggcgat caccgcttcc ctcatactct tcctttttca 1860
atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat 1920
ttagaaaaat aaacaaatag ctagctcact cggtcgctac gctccgggcg tgagactgcg 1980
gcgggcgctg cggacacata caaagttacc cacagattcc gtggataagc aggggactaa 2040
catgtgaggc aaaacagcag ggccgcgccg gtggcgtttt tccataggct ccgccctcct 2100
gccagagttc acataaacag acgcttttcc ggtgcatctg tgggagccgt gaggctcaac 2160
catgaatctg acagtacggg cgaaacccga caggacttaa agatccccac cgtttccggc 2220
gggtcgctcc ctcttgcgct ctcctgttcc gaccctgccg tttaccggat acctgttccg 2280
cctttctccc ttacgggaag tgtggcgctt tctcatagct cacacactgg tatctcggct 2340
cggtgtaggt cgttcgctcc aagctgggct gtaagcaaga actccccgtt cagcccgact 2400
gctgcgcctt atccggtaac tgttcacttg agtccaaccc ggaaaagcac ggtaaaacgc 2460
cactggcagc agccattggt aactgggagt tcgcagagga tttgtttagc taaacacgcg 2520
gttgctcttg aagtgtgcgc caaagtccgg ctacactgga aggacagatt tggttgctgt 2580
gctctgcgaa agccagttac cacggttaag cagttcccca actgacttaa ccttcgatca 2640
aaccacctcc ccaggtggtt ttttcgttta cagggcaaaa gattacgcgc agaaaaaaag 2700
gatctcaaga agatcctttg atcttttcta ctgaaccgct ctagatttca gtgcaattta 2760
tctcttcaaa tgtagcacct gaagtcagcc ccatacgata taagttgtaa ttctcatgtt 2820
agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg 2880
tcgagatccc ggtgcctaat gagtgagcta acttacatta attgcgttgc gctcactgcc 2940
cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg 3000
gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt gagacgggca 3060
acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg 3120
tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata taacatgagc 3180
tgtcttcggt atcgtcgtat cccactaccg agatgtccgc accaacgcgc agcccggact 3240
cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg 3300
gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg gcactccagt 3360
cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta tgccagccag 3420
ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg atttgctggt 3480
gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa 3540
tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca ttagtgcagg 3600
cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc agcccactga 3660
cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta 3720
ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc gccgcgacaa 3780
tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc aacgactgtt 3840
tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc atcgccgctt 3900
ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg 3960
tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt ttcacattca 4020
ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag gttttgcgcc 4080
attcgatggt gtccgggatc tcgacgctct cccttatgcg actcctgcat ta 4132
<210> 4
<211> 7961
<212> DNA
<213> Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 4
tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60
cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 120
ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180
gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240
acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 300
ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 360
ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 420
acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 480
tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 540
tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat 600
gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt 660
ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg 720
agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga 780
agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg 840
tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt 900
tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg 960
cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg 1020
aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga 1080
tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc 1140
tgcagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc 1200
ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc 1260
ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg 1320
cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac 1380
gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc 1440
actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt 1500
aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac 1560
caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa 1620
aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc 1680
accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt 1740
aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg 1800
ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc 1860
agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt 1920
accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga 1980
gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct 2040
tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg 2100
cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca 2160
cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa 2220
cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt 2280
ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 2340
taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 2400
gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatatgg 2460
tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat 2520
cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct 2580
gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct 2640
gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct 2700
catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt 2760
tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg 2820
ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa 2880
tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc 2940
ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa 3000
aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta 3060
gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg 3120
tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag 3180
acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac 3240
cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca 3300
cccgtggcca ggacccaacg ctgcccgaga tctcgatccc gcgaaatctg aaaggaggaa 3360
ctatactcgg atatctgttc acattcgaac cgtctctgct ttgacatctt atgattctcg 3420
actgtaaagt cgtggccaca acgctgcacc cgaatcacat tacggactat tattatgaac 3480
acgattaaca tcgctaagaa cgacttctct gacatcgaac tggctgctat cccgctcaac 3540
actctggctg accattacgg tgagcgttca gctcgcggac agttggccct tgagcatgag 3600
tcttacgaga tgggtgaagc acgcttccgc aagatgtttg agtgtcaact taaagctggt 3660
aaggttgcgg ataacgctgc cgccaagcct ctcatcacta ccctcctccc taagatgatt 3720
gcccgcatca acgactggtt tgaggaagtg aaagctaagc gcggcaggcg cccgacagcc 3780
ttcaagttcc tcaaagaaat caagccggaa gccgtagcgt acatcaccat taagacctct 3840
ctggcttgcc tcaccagtgc tgacaataca accgttcagg ctgtagcaag cgcaatcggt 3900
cggaccattg aggacgaggc tcgcttcggt cgtatccgtg accttgaagc taagcacttc 3960
aagaaaaacg ttgaggaaca actcaacaag cgcgtagggc acgtctacaa gggtggatcc 4020
ggctctggtt cgagttctgt cgctcctgag accgtgggct ctacaagtgt ggtcgcagtg 4080
gtcttcccat ctcacatttt tgtcgcaaat tgcggcgaca gccgggccgt gctgtgcagg 4140
ggaaagaccg ccctgccact gagcgtggac cataaacccg acagggagga tgaagcagcc 4200
cgcatcgagg ctgcaggagg caaagtgatc cagtggaacg gagcacgggt gtttggcgtc 4260
ctggccatgt cacgcagcat tggggatcga tacctgaaac catcaatcat tcccgaccct 4320
gaagtgactg ccgtcaagag agtgaaagaa gacgattgcc tgatcctggc tagcgacggc 4380
gtctgggatg tgatgaccga cgaggaagca tgtgagatgg cccgaaagcg gattctgctg 4440
tggcataaga aaaatgccgt ggctggcgat gctagtctgc tggcagacga gcggagaaag 4500
gaagggaaag atcccgccgc tatgagtgca gccgaatatc tgtcaaaact ggctattcag 4560
aggggcagta aggacaacat ctccgtcgtc gtcgtggacc tgaagggcgg tagtggttcg 4620
ggttcttcgg tccccctgta tgggttcacc agcatttgtg ggcggagacc agagatggag 4680
gcagccgtca gcactattcc ccggttcctg cagtcaagct ccggcagcat gctggacggg 4740
aggttcgatc cacagtccgc cgctcacttc tttggggtct acgatggaca tggcgggtcc 4800
caggtggcca actattgcag ggagcgcatg cacctggctc tggcagagga aatcgccaag 4860
gagaaaccta tgctgtgcga cggagataca tggctggaaa agtggaagaa agccctgttc 4920
aactcttttc tgcgggtgga ctccgagatc gaatgaggta ccattcaaga cccccgcacc 4980
gaaaggtccg ggggtttttt ttactattta aatcctgcca gtgttcacat tcgaaccgtc 5040
tctgctttga caacatgctg tgcggtgttg taaagtcgtg gccaggagaa tacgacaggc 5100
aattgcaaga aggaggatat tgatgccatc ggaacttaca ccggaggaaa ggtccgagtt 5160
gaagaacagc attgcggagt ttcatacata tcaactcgat ccggggagtt gttctagttt 5220
gcacgcccaa aggatacacg ccccaccgga acttgtgtgg agtatcgtcc gccggttcga 5280
taaaccacaa acacacaggc atttcattaa aagctgctcc gtggagcaga actttgaaat 5340
gagagtggga tgcacacggg atataatagt tataagcggc ctcccggcca acacgagcac 5400
cgaaagattg gacatactgg atgacgagcg ccgcgtgacg ggggcatcaa taatcggtgg 5460
ggaacatcgc cttacgaatt ataaaggcgt cactactgtt catagatttg agaaggaaaa 5520
tagaatctgg actgttgttc tggaatctta tgtggttgat atgccagagg gtaactcgga 5580
agatgataca cgcatgctgg cggacacggt tgtgaaactt aatctgcaaa agctggcaac 5640
tgtcgccgaa gccatggcag gtagcgcagg atcaggctcg agtggtaaag catttatgca 5700
agttgtcgag gctgacatgc tctctaaggg tctcctcggt ggcgaggcgt ggtcttcgtg 5760
gcataaggaa gactctattc atgtaggagt acgctgcatc gagatgctca ttgagtcaac 5820
cggaatggtt agcctccacc gccaaaatgc tggcgtagta ggtcaagact ctgagactat 5880
cgaactcgca cctgaatacg ctgaggctat cgcaacccgt gcaggtgcgc tggctggcat 5940
ctctccgatg ttccaacctt gcgtagttcc tcctaagccg tggactggca ttactggtgg 6000
tggctattgg gctaacggtc gtcgtcctct ggcgctggtg cgtactcaca gtaagaaagc 6060
actgatgcgc tacgaagacg tttacatgcc tgaggtgtac aaagcgatta acattgcgca 6120
aaacaccgca tggaaaatca acaagaaagt cctcgcggtc gccaacgtaa tcaccaagtg 6180
gaagcattgt ccggtcgagg acatccctgc gattgagcgt gaagaactcc cgatgaaacc 6240
ggaagacatc gacatgaatc ctgaggctct caccgcgtgg aaacgtgctg ccgctgctgt 6300
gtaccgcaag gacaaggctc gcaagtctcg ccgtatcagc cttgagttca tgcttgagca 6360
agccaataag tttgctaacc ataaggccat ctggttccct tacaacatgg actggcgcgg 6420
tcgtgtttac gctgtgtcaa tgttcaaccc gcaaggtaac gatatgacca aaggactgct 6480
tacgctggcg aaaggtaaac caatcggtaa ggaaggttac tactggctga aaatccacgg 6540
tgcaaactgt gcgggtgtcg ataaggttcc gttccctgag cgcatcaagt tcattgagga 6600
aaaccacgag aacatcatgg cttgcgctaa gtctccactg gagaacactt ggtgggctga 6660
gcaagattct ccgttctgct tccttgcgtt ctgctttgag tacgctgggg tacagcacca 6720
cggcctgagc tataactgct cccttccgct ggcgtttgac gggtcttgct ctggcatcca 6780
gcacttctcc gcgatgctcc gagatgaggt aggtggtcgc gcggttaact tgcttcctag 6840
tgaaaccgtt caggacatct acgggattgt tgctaagaaa gtcaacgaga ttctccaagc 6900
agacgcaatc aatgggaccg ataacgaagt agttaccgtg accgatgaga acactggtga 6960
aatctctgag aaagtcaagc tgggcactaa ggcactggct ggtcaatggc tggcttacgg 7020
tgttactcgc agtgtgacta agcgttcagt catgacgctg gcttacgggt ccaaagagtt 7080
cggcttccgt caacaagtgc tggaagatac cattcagcca gctattgatt ccggcaaggg 7140
tctgatgttc actcagccga atcaggctgc tggatacatg gctaagctga tttgggaatc 7200
tgtgagcgtg acggtggtag ctgcggttga agcaatgaac tggcttaagt ctgctgctaa 7260
gctgctggct gctgaggtca aagataagaa gactggagag attcttcgca agcgttgcgc 7320
tgtgcattgg gtaactcctg atggtttccc tgtgtggcag gaatacaaga agcctattca 7380
gacgcgcttg aacctgatgt tcctcggtca gttccgcctc cagcctacca ttaacaccaa 7440
caaagatagc gagattgatg cacacaaaca ggagtctggt atcgctccta actttgtaca 7500
cagccaagac ggtagccacc ttcgtaagac tgtagtgtgg gcacacgaga agtacggaat 7560
cgaatctttt gcactgattc acgactcctt cggtaccatt ccggctgacg ctgcgaacct 7620
gttcaaagca gtgcgcgaaa ctatggttga cacatatgag tcttgtgatg tactggctga 7680
tttctacgac cagttcgctg accagttgca cgagtctcaa ttggacaaaa tgccagcact 7740
tccggctaaa ggtaacttga acctccgtga catcctcgag tcggacttcg cgttcgcgta 7800
atagcaccac caccaccacc actgagatcc ggctgctaac aaagcccgaa aggaagctga 7860
gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt 7920
cttgaggggt tttttgctga aaggaggaac tatatccgga t 7961
<210> 5
<211> 4251
<212> DNA
<213> Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 5
agatctcgat cccgcgaaat taatacgact cactataggg agaccacaac ggtttccctc 60
tacaaataat tttgtttaac tttaagaagg agatatacat atgcgtaaag gcgaagagct 120
gttcactggt gtcgtcccta ttctggtgga actggatggt gatgtcaacg gtcataagtt 180
ttccgtgcgt ggcgagggtg aaggtgacgc aactaatggt aaactgacgc tgaagttcat 240
ctgtactact ggtaaactgc cagtaccttg gccgactctg gtaacgacgc tgacttatgg 300
tgttcagtgc tttgctcgtt atccggacca catgaagcag catgacttct tcaagtccgc 360
catgccggaa ggctatgtgc aggaacgcac gatttcattt aaggatgacg gcacgtacaa 420
aacgcgtgcg gaagtgaaat ttgaaggcga taccctggta aaccgcattg agctgaaagg 480
cattgacttt aaagaagacg gcaatatcct gggccataag ctggaataca attttaacag 540
ccacaatgtt tacatcaccg ccgataaaca aaaaaatggc attaaagcga atttcaaaat 600
tcgccacaac gtggaggatg gcagcgtgca gctggctgat cactaccagc aaaacactcc 660
aatcggtgat ggtcctgttc tgctgccaga caatcactat ctgagcacgc aaagcgttct 720
gtctaaagat ccgaacgaga aacgcgatca catggttctg ctggagttcg taaccgcagc 780
gggcatcacg cacggtatgg atgaactgta caaatgagaa ttcttgttca gaacgctcgg 840
tcttgcacac cgggcgtttt ttctttgtga gtccaatcca aaacgccgcg ttcagcggcg 900
ttttttctgc ttctgaaagg aggaactata ctcggatatc ccgctgagca ataactagca 960
taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaacc tcaggcattt 1020
gagaagcaca cggtcacact gcttccggta gtcaataaac cggtaaacca gcaatagaca 1080
taagcggcta tttaacgacc ctgccctgaa ccgacgaccg ggtcatcgtg gccggatctt 1140
gcggcccctc ggcttgaacg aattgttaga cattatttgc cgactacctt ggtgatctcg 1200
cctttcacgt agtggacaaa ttcttccaac tgatctgcgc gcgaggccaa gcgatcttct 1260
tcttgtccaa gataagcctg tctagcttca agtatgacgg gctgatactg ggccggcagg 1320
cgctccattg cccagtcggc agcgacatcc ttcggcgcga ttttgccggt tactgcgctg 1380
taccaaatgc gggacaacgt aagcactaca tttcgctcat cgccagccca gtcgggcggc 1440
gagttccata gcgttaaggt ttcatttagc gcctcaaata gatcctgttc aggaaccgga 1500
tcaaagagtt cctccgccgc tggacctacc aaggcaacgc tatgttctct tgcttttgtc 1560
agcaagatag ccagatcaat gtcgatcgtg gctggctcga agatacctgc aagaatgtca 1620
ttgcgctgcc attctccaaa ttgcagttcg cgcttagctg gataacgcca cggaatgatg 1680
tcgtcgtgca caacaatggt gacttctaca gcgcggagaa tctcgctctc tccaggggaa 1740
gccgaagttt ccaaaaggtc gttgatcaaa gctcgccgcg ttgtttcatc aagccttacg 1800
gtcaccgtaa ccagcaaatc aatatcactg tgtggcttca ggccgccatc cactgcggag 1860
ccgtacaaat gtacggccag caacgtcggt tcgagatggc gctcgatgac gccaactacc 1920
tctgatagtt gagtcgatac ttcggcgatc accgcttccc tcatactctt cctttttcaa 1980
tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt 2040
tagaaaaata aacaaatagc tagctcactc ggtcgctacg ctccgggcgt gagactgcgg 2100
cgggcgctgc ggacacatac aaagttaccc acagattccg tggataagca ggggactaac 2160
atgtgaggca aaacagcagg gccgcgccgg tggcgttttt ccataggctc cgccctcctg 2220
ccagagttca cataaacaga cgcttttccg gtgcatctgt gggagccgtg aggctcaacc 2280
atgaatctga cagtacgggc gaaacccgac aggacttaaa gatccccacc gtttccggcg 2340
ggtcgctccc tcttgcgctc tcctgttccg accctgccgt ttaccggata cctgttccgc 2400
ctttctccct tacgggaagt gtggcgcttt ctcatagctc acacactggt atctcggctc 2460
ggtgtaggtc gttcgctcca agctgggctg taagcaagaa ctccccgttc agcccgactg 2520
ctgcgcctta tccggtaact gttcacttga gtccaacccg gaaaagcacg gtaaaacgcc 2580
actggcagca gccattggta actgggagtt cgcagaggat ttgtttagct aaacacgcgg 2640
ttgctcttga agtgtgcgcc aaagtccggc tacactggaa ggacagattt ggttgctgtg 2700
ctctgcgaaa gccagttacc acggttaagc agttccccaa ctgacttaac cttcgatcaa 2760
accacctccc caggtggttt tttcgtttac agggcaaaag attacgcgca gaaaaaaagg 2820
atctcaagaa gatcctttga tcttttctac tgaaccgctc tagatttcag tgcaatttat 2880
ctcttcaaat gtagcacctg aagtcagccc catacgatat aagttgtaat tctcatgtta 2940
gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt 3000
cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc 3060
gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg 3120
agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa 3180
cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt 3240
ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct 3300
gtcttcggta tcgtcgtatc ccactaccga gatgtccgca ccaacgcgca gcccggactc 3360
ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg 3420
aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc 3480
gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc 3540
cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg 3600
acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat 3660
actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc 3720
agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac 3780
gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac 3840
catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat 3900
ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt 3960
gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc 4020
cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt 4080
ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac 4140
caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca 4200
ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt a 4251
<210> 6
<211> 537
<212> DNA
<213> Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 6
atgaacacga ttaacatcgc taagaacgac ttctctgaca tcgaactggc tgctatcccg 60
ctcaacactc tggctgacca ttacggtgag cgttcagctc gcggacagtt ggcccttgag 120
catgagtctt acgagatggg tgaagcacgc ttccgcaaga tgtttgagtg tcaacttaaa 180
gctggtaagg ttgcggataa cgctgccgcc aagcctctca tcactaccct cctccctaag 240
atgattgccc gcatcaacga ctggtttgag gaagtgaaag ctaagcgcgg caggcgcccg 300
acagccttca agttcctcaa agaaatcaag ccggaagccg tagcgtacat caccattaag 360
acctctctgg cttgcctcac cagtgctgac aatacaaccg ttcaggctgt agcaagcgca 420
atcggtcgga ccattgagga cgaggctcgc ttcggtcgta tccgtgacct tgaagctaag 480
cacttcaaga aaaacgttga ggaacaactc aacaagcgcg tagggcacgt ctacaag 537
<210> 7
<211> 918
<212> DNA
<213> Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 7
tctgtcgctc ctgagaccgt gggctctaca agtgtggtcg cagtggtctt cccatctcac 60
atttttgtcg caaattgcgg cgacagccgg gccgtgctgt gcaggggaaa gaccgccctg 120
ccactgagcg tggaccataa acccgacagg gaggatgaag cagcccgcat cgaggctgca 180
ggaggcaaag tgatccagtg gaacggagca cgggtgtttg gcgtcctggc catgtcacgc 240
agcattgggg atcgatacct gaaaccatca atcattcccg accctgaagt gactgccgtc 300
aagagagtga aagaagacga ttgcctgatc ctggctagcg acggcgtctg ggatgtgatg 360
accgacgagg aagcatgtga gatggcccga aagcggattc tgctgtggca taagaaaaat 420
gccgtggctg gcgatgctag tctgctggca gacgagcgga gaaaggaagg gaaagatccc 480
gccgctatga gtgcagccga atatctgtca aaactggcta ttcagagggg cagtaaggac 540
aacatctccg tcgtcgtcgt ggacctgaag ggcggtagtg gttcgggttc ttcggtcccc 600
ctgtatgggt tcaccagcat ttgtgggcgg agaccagaga tggaggcagc cgtcagcact 660
attccccggt tcctgcagtc aagctccggc agcatgctgg acgggaggtt cgatccacag 720
tccgccgctc acttctttgg ggtctacgat ggacatggcg ggtcccaggt ggccaactat 780
tgcagggagc gcatgcacct ggctctggca gaggaaatcg ccaaggagaa acctatgctg 840
tgcgacggag atacatggct ggaaaagtgg aagaaagccc tgttcaactc ttttctgcgg 900
gtggactccg agatcgaa 918
<210> 8
<211> 537
<212> DNA
<213> Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 8
atgccatcgg aacttacacc ggaggaaagg tccgagttga agaacagcat tgcggagttt 60
catacatatc aactcgatcc ggggagttgt tctagtttgc acgcccaaag gatacacgcc 120
ccaccggaac ttgtgtggag tatcgtccgc cggttcgata aaccacaaac acacaggcat 180
ttcattaaaa gctgctccgt ggagcagaac tttgaaatga gagtgggatg cacacgggat 240
ataatagtta taagcggcct cccggccaac acgagcaccg aaagattgga catactggat 300
gacgagcgcc gcgtgacggg ggcatcaata atcggtgggg aacatcgcct tacgaattat 360
aaaggcgtca ctactgttca tagatttgag aaggaaaata gaatctggac tgttgttctg 420
gaatcttatg tggttgatat gccagagggt aactcggaag atgatacacg catgctggcg 480
gacacggttg tgaaacttaa tctgcaaaag ctggcaactg tcgccgaagc catggca 537
<210> 9
<211> 2115
<212> DNA
<213> Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 9
aaagcattta tgcaagttgt cgaggctgac atgctctcta agggtctcct cggtggcgag 60
gcgtggtctt cgtggcataa ggaagactct attcatgtag gagtacgctg catcgagatg 120
ctcattgagt caaccggaat ggttagcctc caccgccaaa atgctggcgt agtaggtcaa 180
gactctgaga ctatcgaact cgcacctgaa tacgctgagg ctatcgcaac ccgtgcaggt 240
gcgctggctg gcatctctcc gatgttccaa ccttgcgtag ttcctcctaa gccgtggact 300
ggcattactg gtggtggcta ttgggctaac ggtcgtcgtc ctctggcgct ggtgcgtact 360
cacagtaaga aagcactgat gcgctacgaa gacgtttaca tgcctgaggt gtacaaagcg 420
attaacattg cgcaaaacac cgcatggaaa atcaacaaga aagtcctcgc ggtcgccaac 480
gtaatcacca agtggaagca ttgtccggtc gaggacatcc ctgcgattga gcgtgaagaa 540
ctcccgatga aaccggaaga catcgacatg aatcctgagg ctctcaccgc gtggaaacgt 600
gctgccgctg ctgtgtaccg caaggacaag gctcgcaagt ctcgccgtat cagccttgag 660
ttcatgcttg agcaagccaa taagtttgct aaccataagg ccatctggtt cccttacaac 720
atggactggc gcggtcgtgt ttacgctgtg tcaatgttca acccgcaagg taacgatatg 780
accaaaggac tgcttacgct ggcgaaaggt aaaccaatcg gtaaggaagg ttactactgg 840
ctgaaaatcc acggtgcaaa ctgtgcgggt gtcgataagg ttccgttccc tgagcgcatc 900
aagttcattg aggaaaacca cgagaacatc atggcttgcg ctaagtctcc actggagaac 960
acttggtggg ctgagcaaga ttctccgttc tgcttccttg cgttctgctt tgagtacgct 1020
ggggtacagc accacggcct gagctataac tgctcccttc cgctggcgtt tgacgggtct 1080
tgctctggca tccagcactt ctccgcgatg ctccgagatg aggtaggtgg tcgcgcggtt 1140
aacttgcttc ctagtgaaac cgttcaggac atctacggga ttgttgctaa gaaagtcaac 1200
gagattctcc aagcagacgc aatcaatggg accgataacg aagtagttac cgtgaccgat 1260
gagaacactg gtgaaatctc tgagaaagtc aagctgggca ctaaggcact ggctggtcaa 1320
tggctggctt acggtgttac tcgcagtgtg actaagcgtt cagtcatgac gctggcttac 1380
gggtccaaag agttcggctt ccgtcaacaa gtgctggaag ataccattca gccagctatt 1440
gattccggca agggtctgat gttcactcag ccgaatcagg ctgctggata catggctaag 1500
ctgatttggg aatctgtgag cgtgacggtg gtagctgcgg ttgaagcaat gaactggctt 1560
aagtctgctg ctaagctgct ggctgctgag gtcaaagata agaagactgg agagattctt 1620
cgcaagcgtt gcgctgtgca ttgggtaact cctgatggtt tccctgtgtg gcaggaatac 1680
aagaagccta ttcagacgcg cttgaacctg atgttcctcg gtcagttccg cctccagcct 1740
accattaaca ccaacaaaga tagcgagatt gatgcacaca aacaggagtc tggtatcgct 1800
cctaactttg tacacagcca agacggtagc caccttcgta agactgtagt gtgggcacac 1860
gagaagtacg gaatcgaatc ttttgcactg attcacgact ccttcggtac cattccggct 1920
gacgctgcga acctgttcaa agcagtgcgc gaaactatgg ttgacacata tgagtcttgt 1980
gatgtactgg ctgatttcta cgaccagttc gctgaccagt tgcacgagtc tcaattggac 2040
aaaatgccag cacttccggc taaaggtaac ttgaacctcc gtgacatcct cgagtcggac 2100
ttcgcgttcg cgtaa 2115
<210> 10
<211> 60
<212> DNA
<213> Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 10
atggacctgc ctgacgacca ctacctgtcc acccagacca tcctgtccaa ggacctgaac 60
<210> 11
<211> 63
<212> DNA
<213> Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 11
gaaaagcgag accatatggt tttgcttgag tatgttacag cggctggcat taccgatgca 60
tca 63
<210> 12
<211> 588
<212> DNA
<213> Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 12
atgcgcaaag gcgaagaact gtttaccggc attgtgccga ttctggtgga actggatggc 60
gatgtgaacg gccataaatt ttttgtgcgc ggcgaaggcg aaggcgatgc gaccattggc 120
aaactgagcc tgaaatttat ttgcaccacc ggcaaactgc cggtgccgtg gccgaccctg 180
gtgaccaccc tgacctatgg cgtgcagtgc tttagccgct atccggatca tatgaaacgc 240
catgattttt ttaaaagcgc gatgccggaa ggctatgtgc aggaacgcac catttatttt 300
aaagatgatg gcacctataa aacccgcgcg gaagtgaaat ttgaaggcga taccctggtg 360
aaccgcattg aactgaaagg cattgatttt aaagaagatg gcaacattct gggccataaa 420
ctggaatata actttaacag ccataaagtg tatattaccg cggataaaca gaacaacggc 480
attaaagcga actttaccat tcgccataac gtggaagatg gcagcgtgca gctggcggat 540
cattatcagc agaacacccc gattggcgat ggcccggttc ttcttcct 588
<210> 13
<211> 87
<212> DNA
<213> Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 13
taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt 60
tgtttaactt taagaaggag atatacc 87
<210> 14
<211> 19
<212> DNA
<213> Artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 14
taatacgact cactatagg 19
Claims (10)
1. A method for pesticide induced protein interaction and induced gene expression, characterized in that: the pesticide is mandipropamid, the induction proteins are mutually complementary with three fragments of the induction green fluorescent protein, the induction gene expression is the induction green fluorescent protein coding gene expression, and the steps are as follows:
step 1: construction of plasmid 2 on the basis of plasmid 1, ABI-CP amino-terminal fusion GFP10, PYR1 MANDI The carboxyl end is fused with GFP11;
step 2: constructing a plasmid 3, and controlling induction expression of GFP1-9 fragments in escherichia coli by using a T7 promoter_lacO to match with an IPTG induction system of escherichia coli BL21 (DE 3) strain;
step 3: transforming the plasmids 2 and 3 into an escherichia coli BL21 (DE 3) strain;
step 4, detecting that the expression products of plasmids 2 and 3 are induced by mandipropamid in E.coli: taking positive clone shake bacteria of escherichia coli BL21 (DE 3) strain background, and waiting for OD 600 Reaching 0.40-0.60, adding IPTG (isopropyl thiogalactoside) with a final concentration of 1mM to the bacterial liquid to induce GFP1-9 fragment expression, and simultaneously adding mandipropamid with a final concentration gradient of 1 mu M,10 mu M and 100 mu M;
step 5: after 3-4 hours, the fluorescence intensities of the three fragments GFP1-9, GFP10 and GFP11 recombined into GFP were measured with the following parameters, excitation light 488nm, emission light 510nm, and OD was measured simultaneously 600 The method is used for homogenizing the culture concentration of the bacterial liquid, and the result shows that mandipropamid efficiently induces the complementation of three fragments of green fluorescent protein;
step 6: construction of plasmid 4, resolved T7 RNA polymerase and PYR1 in E.coli MANDI Respectively fusion-expressing with ABI-CP;
step 7: constructing a plasmid 5, driving GFP coding gene expression by using a T7 promoter, and detecting the activity of mandipropamid-induced T7 RNA polymerase in escherichia coli;
step 8: transforming the plasmids 4 and 5 into an escherichia coli Top10 strain;
step 9, detection of expression products of plasmids 4 and 5 induced by mandipropamid in E.coli: taking positive clone shake bacteria of escherichia coli Top10 strain background, and waiting for OD 600 Reaching 0.40-0.60, adding mandipropamid with final concentration gradient of 0.1 μm,1 μm,10 μm,100 μm,200 μm,400 μm into bacterial liquid, detecting GFP expression effect with excitation light 488nm and emission light 510nm after 3-4 hr, and simultaneously determining OD 600 For homogenizing the culture concentration of the bacterial liquid, and as a resultShowing that mandipropamid induces the expression of a green fluorescent protein coding gene;
the sequence of plasmid 1 is shown as SEQ ID NO.1, the sequence of plasmid 2 is shown as SEQ ID NO.2, the sequence of plasmid 3 is shown as SEQ ID NO.3, the sequence of plasmid 4 is shown as SEQ ID NO.4, the sequence of plasmid 5 is shown as SEQ ID NO.5, the sequence of T7N is shown as SEQ ID NO.6, and the sequence of ABI1-CP is shown as SEQ ID NO.7, PYR1 MANDI The sequence is shown as SEQ ID NO.8, the T7C sequence is shown as SEQ ID NO.9, the GFP10 sequence is shown as SEQ ID NO.10, the GFP11 sequence is shown as SEQ ID NO.11, the GFP1-9 sequence is shown as SEQ ID NO.12, the T7 promoter_lacO sequence is shown as SEQ ID NO.13, and the T7 promoter sequence is shown as SEQ ID NO. 14.
2. The method of pesticide induced protein interaction and induced gene expression according to claim 1 wherein: the plasmid 1 contains two expression cassettes, and PYR1 is respectively expressed in the escherichia coli in a constitutive mode MANDI And ABI-CP.
3. The method of pesticide induced protein interaction and induced gene expression according to claim 1 wherein: plasmid 2, constructed based on plasmid 1, contains two expression cassettes, ABI-CP expressing amino-terminal fusion GFP10 fragment and PYR1 of carboxyl-terminal fusion GFP11 in E.coli respectively and constitutively MANDI 。
4. The method of pesticide induced protein interaction and induced gene expression according to claim 1 wherein: the plasmid 3 was induced to express GFP1-9 fragment in E.coli by IPTG (isopropyl thiogalactoside).
5. The method of pesticide induced protein interaction and induced gene expression according to claim 1 wherein: the plasmid 4, constructed based on plasmid 1, contains two expression cassettes, which allow the split T7 RNA polymerase and PYR1 in E.coli MANDI And ABI-CP are respectively fused and expressed.
6. The method of pesticide induced protein interaction and induced gene expression according to claim 1 wherein: the expression frame of the plasmid 5 contains a T7 promoter to drive the target gene to express, and the activity of T7 RNA polymerase after induction of mandipropamid is detected in escherichia coli.
7. The method of pesticide induced protein interaction and induced gene expression according to claim 1 wherein: the concentration gradient of the mandipropamid in the step 4 is 1 mu M,10 mu M and 100 mu M, and the concentration gradient of the mandipropamid in the step 9 is 0.1 mu M,1 mu M,10 mu M,100 mu M,200 mu M and 400 mu M.
8. The method of pesticide induced protein interaction and induced gene expression according to claim 1 wherein: the strain with endogenous T7 RNA polymerase cannot be used in step 8.
9. The method for pesticide induced protein interactions and induced gene expression according to claim 8 wherein: the strain with the endogenous T7 RNA polymerase is BL21 (DE 3) strain.
10. The method of pesticide induced protein interaction and induced gene expression according to claim 1 wherein: the positive clone strain in the step 4 is BL21 (DE 3), and the positive clone strain in the step 9 is Top10.
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| CN108410909A (en) * | 2018-04-12 | 2018-08-17 | 吉林大学 | A method of adjusting cell pathway using plant hormone ABA and small-molecule substance PYR |
| CN111979166A (en) * | 2020-08-14 | 2020-11-24 | 中国环境科学研究院 | Engineering bacterium for specifically removing arsenic as well as construction method and application thereof |
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|---|---|---|---|---|
| CN108410909A (en) * | 2018-04-12 | 2018-08-17 | 吉林大学 | A method of adjusting cell pathway using plant hormone ABA and small-molecule substance PYR |
| CN111979166A (en) * | 2020-08-14 | 2020-11-24 | 中国环境科学研究院 | Engineering bacterium for specifically removing arsenic as well as construction method and application thereof |
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| PYR/PYL/RCAR family members are major in-vivo ABI1 protein phosphatase 2C-interacting proteins in Arabidopsis;NORIYUKI,N. 等;《The Plant Journal》;20101231;290-299 * |
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