CN108504679A - The recombination Arthobotrys oligospora and preparation method thereof of Aoz1 gene double-promoters - Google Patents
The recombination Arthobotrys oligospora and preparation method thereof of Aoz1 gene double-promoters Download PDFInfo
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Abstract
A kind of recombination Arthobotrys oligospora and preparation method thereof of the Aoz1 gene double-promoters of ruminant digestion road nematodiasis the invention discloses prevent caused by Nemathelminthes Nematoda, the present invention has cloned Arthobotrys oligospora Aoz1 gene promoters by using chromosomal DNA step shifting technology, and the recombination Arthobotrys oligospora of high efficient expression Aoz1 gene double-promoters is constructed using Agrobacterium tumefaciens mediated fungal transformation technology.Compared with conventional genetic method for transformation, method acceptor material range of the invention is wide, transformation efficiency is high, transformant stablizes the advantages that hereditary, overcomes the bottleneck of Conventional reformat method.Compared with the source of parents Arthobotrys oligospora strain of non-recombinant, the bacterial strain that the present invention is obtained has good genetic stability, its Aoz1 gene transcription level enhances, nematode-trapping activity improves, and the research and development for the anti-controlling agent of ruminant digestion road nematodiasis biology provide an efficient engineering strain.
Description
Technical field
The present invention relates to a kind of Arthobotrys oligospora serine protease Aoz1 gene double-promoters to recombinate few spore Arthrobotrys
Bacterium and preparation method thereof belongs to biotechnology.
Technical background
Ruminant digestion road nematodiasis is that the various nematosises belonging to Nemathelminthes Nematoda disappear in ruminant
Change a kind of parasitic disease caused by road, seriously endangers the sustainable and healthy development of aquaculture industry of China.According to statistics, in ruminant
In verminosis, metenteron line insect infection accounts for more than half, belongs to nematode, blood lance category nematode, Nematodirus nematode, Austria wherein justifying with hair
Si Te belongs to nematode, Marshallagia nematode, based on hair tail category nematode.In recent years, as country is to zoonosis prevention and control measure
Constantly reinforce, the infectious disease of ruminant is controlled effectively, and the harm of parasitic disease becomes increasingly conspicuous.For a long time, right
The prevention of ruminant digestion road nematodiasis relies primarily on chemical anthelmintic, however the prolonged application of chemicals causes polypide resistance to
The generation of pharmacological property so that original efficient chemical anthelmintic drug effect constantly declines, and can only carry out expelling parasite by increasing dose, lead
The problems such as causing the pollution to environment of medicament residue in the animal foods such as beef and mutton and drug metabolite getting worse, gives people
Health bring serious threat.Therefore, in order to overcome chemical expelling parasite medical treatment ruminant digestion road nematodiasis institute band
The drawback come, there is an urgent need to seek a kind of method of new prevention and control ruminant nematodiasis at present.
Numerous research confirms that it is that prevention and control are anti-to carry out biological control using natural enemy-nematode-destroying fungus of nematode
A kind of safely and effectively method of hay animal alimentary canal nematodiasis.
Gronvold etc. (1993), which has carried out, utilizes nematode-destroying fungus-Arthobotrys oligospora (Arthrobotrys
Oligospora) domestic animal cooperid (Cooperia oncophor) infective larvae is expanded from excrement to grassland environment around
Scattered inhibition research, as a result, it has been found that grassland infectivity nematode larval reduces around Arthobotrys oligospora effect group excrement
96.2%.Larsen etc. (1993) confirms that addition Arthobotrys oligospora and Duddingtonia belonged in cow dung catches by testing
85% the 3rd phase larva of ostertagi can be killed after food nematode property fungi.Chandrawathani etc. (1998) utilizes few spore
Arthrobotrys bacterium has carried out laboratory excrement desinsection to the strongyloides papillosus (Strongyloides papillosus) in cow dung
Experiment, as a result 99% nematode larval is killed.In recent decades, Denmark, Britain, Australia and U.S. etc. carry out and use in succession
Nematode-destroying fungus carries out animal alimentary canal parasitic disease the research of biological prevention and control, has filtered out suitable national conditions, work
With good bacterial strain, and animal alimentary canal nematodiasis biology prevention and control experiment has been carried out in production practice, achieve ideal effect
Fruit.Wherein, the international monopoly using nematode-destroying fungus prevention and control animal alimentary canal nematodiasis has been declared by Denmark, and carries out
The R＆D and promotion of the anti-controlling agent of commercialization biology, achieves ideal effect.
According to predacious organs type, nematode-destroying fungus is divided into two kinds, and one is sets to catch type nematode-destroying fungus, represents
Kind is Arthobotrys oligospora (Arthrobotrys oligospora), can generate the predacious organs such as collarium, slime bacteria net, is caught with set
Mode nematode-trapping larva.Another kind is to glue to catch type nematode-destroying fungus, and representative species is Dactylella ellipsospora (Dactylella
Ellipsospora), the predacious organs such as bacterium knot can be formed, the nematode-trapping larva in a manner of viscous catch.Nematode-destroying fungus is caught
Food process such as can be divided into attraction, identify and stick, infect, clearing up at the processes.Nematode-destroying fungus exists first in nematode-trapping
The mycelia of nematode-destroying fungus forms predation structure (predation ring or predation net etc.) under nematode-inducible, and predation structure attracts host
Simultaneously with nematode specific recognition occurs for nematode, and nematode is entangled or adhered, while nematode-destroying fungus secretes some ectoenzymes
Hydrolase promotes mycelia to be directed through nematode body wall, to kill nematode.
As a kind of typical nematode-destroying fungus, Arthobotrys oligospora (Arthrobotrys oligospora) energy
It is enough to generate specific predation structure-stickiness bacterium net to capture nematode, and generate in infecting nematode processes serine protease,
The extracellular hydrolase such as chitinase and clostridiopetidase A completes consolidating for nematode under predation structure and the collective effect of extracellular hydrolase
The fixed, intrusion of cuticula and the degradation of nematode polypide, therefore extracellular hydrolase is in the process of Arthobotrys oligospora nematode-trapping
In played important function.In the extracellular hydrolase of Arthobotrys oligospora secretion, serine protease (serine
Proteases) be it is a kind of it is important infect nematode enzyme, which has played important function in preying on, infecting the nematode stage.
Tunlid etc. (1994) by measure Arthobotrys oligospora fluid nutrient medium supernatant hydrolyze Azocoll (azo dyes class it is aobvious
Color substrate) activity, it was demonstrated that the bacterium can generate several different extracellular proteases, including more serine protease,
The activity of these extracellular proteases can be inhibited by phenylmethylsulfonyl fluoride (PMSF) and other several serpins, portion
Divide protease can also be by asparaginic acid protease inhibitors (such as pepstatin) and cystatin (E-64)
Inhibited.Currently, the serine proteases such as PII, Aoz1, P32, VCP1, PL are had discovered that from Arthobotrys oligospora, it
Have very high homology with the member of subtilopeptidase A family, these extracellular proteases can degrade nematode cutin membrane or
Person's egg capsule shell.Zhang etc. (2004) carries out A.oligospora serine protease Aoz1 genes using Reverse Genetics
Clone, gene open reading frame (ORF) the overall length 1281bp encode 426 amino acid, wherein the 1st~21 amino acids are
Aoz1 signal peptides, the amino acid sequence homology with the PII protease for the Arthobotrys oligospora delivered in GenBank are
97.67%, the homology with neutral serine amino acid sequence is 97.89%.
In view of the serious harm of ruminant nematodiasis and increasingly showing especially for chemical anthelmintic drawback, seek a kind of new height
Effect prevention and control ruminant digestion road nematodiasis has great importance.However, the Arthobotrys oligospora detached from nature exists
Although separation initial stage shows stronger nematode-trapping activity, but with the increase in culture medium passage number, extracellular proteolysis
The expression of enzyme also reduces, and nematode-trapping activity can be gradually reduced, this has become restriction and researches and develops efficient nematode-trapping biology
The bottleneck of preparation.
It is to solve to carry out molecular genetic transformation to the Arthobotrys oligospora strain detached in nature using genetic engineering means
One of effective way of the bottleneck.Agrobacterium tumefaciens mediated fungal transformation technology (ATMT) is the weight of strain molecular genetic manipulation
Want one of method.Agrobacterium is Gram-negative agrobacterium, in natural conditions, can be produced by micro- wound infection plant
Raw goiter and tumor, and T-DNA is integrated on plant chromosome, foreign gene is inserted into the areas T-DNA in laboratory conditions, uses T-
The characteristic of DNA conversions obtains the recombinant plant of expression alien gene.Agrobacterium tumefaciens mediated genetic of fungi conversion (ATMT) with
Mediated plant genetic transformation mechanism is similar.ATMT is by both transition range (areas T-DNA) and virulence region (areas Vir) collective effects
It completes.Virulence region is made of a series of Vir genes, the coding albumen of these Vir genes take part in T-DNA formed, transfer and whole
The process of conjunction.(such as PEG protoplast transformations, lithium acetate transformation, electroporated method and restriction enzyme are situated between with conventional genetic method for transformation
Lead conversion) it compares, ATMT has many advantages, such as that acceptor material range is wide, transformation efficiency is high, transformant inheritance stability, becomes to filiform
Fungal bacterial strain carries out one of the important method of genetic modification.
Invention content
The object of the present invention is to provide the ruminant digestion roads a kind of prevent caused by Nemathelminthes Nematoda
The recombination Arthobotrys oligospora and preparation method thereof of the Aoz1 gene double-promoters of nematodiasis.
The present invention has cloned Arthobotrys oligospora Aoz1 gene promoters by using chromosomal DNA step shifting technology, utilizes
Spore section is lacked in the recombination that Agrobacterium tumefaciens mediated fungal transformation technology (ATMT) constructs high efficient expression Aoz1 gene double-promoters
Clump spore bacterium.
The present invention walks shifting technology by chromosomal DNA first and clones Arthobotrys oligospora serine protease Aoz1 gene 5 's
Upstream sequence is held, the gene promoter sequence is transcribed using EGFP promoters report carrier and DNA gel block technique
Active function is identified.On this basis, Aoz1 genes are constructed using Agrobacterium tumefaciens mediated fungal transformation technology (ATMT)
The recombination Arthobotrys oligospora of double-promoter, passes through the effect to haemonchus contortus, it was demonstrated that the Aoz1 gene double startups of structure
The recombination Arthobotrys oligospora of son has good genetic stability, the enhancing of Aoz1 gene transcription levels, nematode-trapping activity
It improves, the development for the anti-controlling agent of ruminant digestion road nematodiasis biology provides an efficient genetic engineering candidate bacterium
Strain.
The present invention represents bacterium-Arthobotrys oligospora as object using nematode-destroying fungus, is walked by chromosomal DNA and moves skill
Art expands Arthobotrys oligospora serine protease Aoz1 gene 5 's ends upstream sequence, using EGFP promoters report carrier and
DNA gel block technique carries out transcriptional activity Function Identification to the gene promoter sequence；Using Agrobacterium tumefaciens mediated true
Bacterium transformation technology (ATMT) constructs the recombination Arthobotrys oligospora of Aoz1 gene double-promoters, and verification Aoz1 gene promoters are double
The recombination Arthobotrys oligospora nematode-trapping activity of promoter, is the development of the anti-controlling agent of ruminant digestion road nematodiasis biology
Provide an efficient genetic engineering candidate strain.
The present invention mainly includes the content of the following aspects：
1, the culture of Arthobotrys oligospora.
2, the clone of Arthobotrys oligospora Aoz1 gene 5 's end upstream sequence and sequence signature analysis.
3, the identification of Arthobotrys oligospora Aoz1 gene promoter sequences and transcriptional activity analysis.
4, the structure of the recombination Arthobotrys oligospora of Aoz1 genes double-promoter.
5, Aoz1 genes double-promoter recombination Arthobotrys oligospora Aoz1 gene expression doses detection.
6, Aoz1 genes double-promoter recombination Arthobotrys oligospora genetic stability analysis.
7, Aoz1 genes double-promoter recombinates Arthobotrys oligospora nematode-trapping activity analysis.
The specific implementation process is as follows：
1, the culture of Arthobotrys oligospora.
Existing Arthobotrys oligospora conidium is taken to be inoculated on corn meal agar culture medium (CMA), 20~30 DEG C of temperature
(Fig. 1) is cultivated in case.The mycelia fritter for cutting above-mentioned culture is put into the niblet triangular flask of sterilizing, and 20~30 DEG C of cultures 18~
25d, with aseptic distillation water elution conidium (Fig. 2), Cord blood is spare after counting.
2, the clone of Arthobotrys oligospora Aoz1 gene 5 's end upstream sequence and sequence signature analysis.
1. the design of primers of Arthobotrys oligospora GenomeWalker.
According to the adapter-primer sequence AP1 in existing kit：5 '-GTAATACGACTCACTATAGGGC-3' and AP2：
5’-ACTATAGGGCACGCGTGGT-3'；It holds end sequence as template using the cDNA 5 ' of Aoz1 genes, designs it
The specific primer of GenomeWalker：GSP1：5 ' end-CGGATTGGGCCAGCAAAGGCATTGGTAGC-3'；GSP2：5'
End-GATTGCTAGAAGGGAGATGAGGCCGTTCG-3'(tables 1).
2. genomic DNA digestion and purifying.
The Arthobotrys oligospora conidium after culture is taken, is ground in the mortar that liquid nitrogen is added, is extracted and is tried with fungal DNA
Agent box extracts Arthobotrys oligospora conidium DNA.With restriction enzyme DraI, EcoRV, PvuII, StuI respectively to few spore
Arthrobotrys bacterium DNA carries out digestion.It is right as the positive simultaneously with the genomic DNA of the mankind provided in PvuII cleavage reagent boxes
According to.With DNA Purification Kit digestion products, DNA is dissolved in 15~25 μ L TE solution.
3. the connection of genomic DNA fragment and connector.
Learn from else's experience four kinds of endonuclease digestions and Arthobotrys oligospora genomic DNA fragment after purification and positive control-mankind
DNA-PvuII digestion fragments are connected with GenomeWalker connectors (Adaptor) respectively.
4. Arthobotrys oligospora Aoz1 gene 5 's end upstream sequence PCR amplification.
Carry out twice PCR reaction：Initial p CR (primary PCR) and secondary PCR (secondary PCR).Initial p CR
It using Arthobotrys oligospora genomic DNA fragment as template, is expanded using AP1-GSP1 primers, second of PCR is with initial p CR
Amplified production is template, is expanded using AP2-GSP2 primers.
Each PCR reactions all set negative control and positive control.PCR system is detected with the positive control that kit provides
Feasibility.As a result it has been obtained on the Arthobotrys oligospora Aoz1 gene 5 's end that size is about 2050bp by second of PCR amplification
Swim genetic fragment (Fig. 3).
5. the bioinformatic analysis of Aoz1 gene 5 's end upstream sequence.
After purification by PCR product, pMD-18T carriers are cloned into, construction recombination plasmid pT-PAoz1 is sequenced, and will be surveyed
Fixed sequence carries out sequence alignment and analysis with Blast softwares on GenBank, with eukaryocyte promoter Analysis software
(Promoter Scan, Promoter Finder and Promoter Prediction Server) analyzes the sequence cis regulatory
The features such as element [TATA-box, GC-box, transcription initiation site Initiator, BRE (TFIIB Recognition
Element)、DPE(Downstream core Promoter Element)：Downstream promoter Conserved Elements, UPE
(Upstream Promoter Element)]。
As a result clone has obtained the Aoz1 gene 5 's end upstream sequence of 2041bp, i.e.,<210>1.
Bioinformatic analysis discovery is carried out, finds that there are TATA box, CAAT in the upstream sequence of Aoz1 gene 5 's end
Box, GC box and the islands CpG, also contain Tubby, ZnCys_Ab0079, NF-X1_Pbl0001, Sp1, GCF, PEBP2, EIIF,
The binding site of the transcription factors such as element_II_rs-4, E2F, EivF/CREB.
3, Arthobotrys oligospora Aoz1 gene promoter transcriptions Activity determination.
1. the structure of the promoter expression vector containing EGFP reporter genes.
By pPIC9K-EGFP promoter detections carrier SacI and BamHI double digestions, cuts off 5 ' AOX1 on carrier and start
Son recycles carrier large fragment.By recombinant plasmid pT-PAoz1 SacI and BamHI double digestions, gel extraction Aoz1 bases after electrophoresis
Because of promoter PAoz1 segments, by the Aoz1 gene promoter PAoz1 segments of recycling and pPIC9K-EGFP promoter detection carriers
Segment overnight, is converted 80~120 μ L Top10 competent cells, utilizes the bacterium solution side PCR with 14~18 DEG C of connections of T4DNA ligases
Method, which is selected, obtains recombinant promoter Expression vector pPIC9K-PAoz1-EGFP positive colonies.
2. recombinant vector converts.
The aseptic double-distilled water that the Arthobotrys oligospora conidium of preparation is pre-chilled is resuspended, then molten with the sorbierite of precooling
Liquid washs conidium, and finally bacterial sediment is resuspended with sorbitol solution, obtains Arthobotrys oligospora conidium competence
Cell.By the Arthobotrys oligospora Aoz1 gene promoter report carriers of pPIC9K-EGFP (empty vectors) and structure
(pPIC9K-PAoz1-EGFP) electrotransformation Arthobotrys oligospora point is distinguished under conditions of 1-2kv, 15-25 μ F and 150-250 Ω
Raw spore.After electricity turns, it is added the culture medium of precooling in the cup that shocks by electricity, and by conidium inoculation method corn meal agar culture
It is cultivated on base tablet.
3. the transcription and translation level of reporter gene EGFP detects.
Take respectively pPIC9K-EGFP (blank control group) and with the conidiums of pPIC9K-Aoz1-EGFP processing groups from
The heart after being resuspended with fixer, stands 1h at room temperature.Dyeing liquor is added to dye 4-6h at 36~38 DEG C, 70~80% ethanol decolorizations,
Northern blot detections are carried out with the DIG EGFP gene nucleic acid probes marked.As a result the EGFP gene nucleic acid marked with DIG
Probe can detect the transcription (mRNA) of EGFP gene；It can detect that band conidium sends out yellow green with fluorescence microscope
Fluorescence (Fig. 4).
4. Aoz1 gene promoter DNA gel retardation experiments.
According to the Aoz1 gene 5 's end upstream sequence design probe (DePAoz1) of acquisition：5 ' ends-
GGGATACGTACGTGCTTAACTCCGCAATCCGGATGGGATCTTG-3', synthesising probing needle in vitro, with gel blocking reagent
5 ' end of probe DePAoz1 sequences is marked in box (DIG Gel Shift Kit), obtains required nucleic acid probe, uses
Ultraviolet specrophotometer measures DNA concentration and purity, the Nuclear extract extracts kit extraction for then using Sigma companies to produce
Conidial nucleoprotein.By the probe and the careful mixing of nucleoprotein of DIG labels, 15~25 DEG C of placement 15min；4~6uL is added
Sample loading buffer carries out polyacrylamide gel electrophoresis, and electrophoresis to blue bands is at bottom 2/3.By the Buddhist nun with gel size
Imperial film filter paper, which is placed in 0.5 × TBE, balances 5min, carries out transferring film and fixation.Nylon membrane (DNA is face-up) is placed on hybridization bag
In, add 1mL CSPD working solutions, closes hybridization bag, 36~38 DEG C of incubation 30min.X-ray film is exposed the film to, chemical hair is carried out
Light detection.As a result Aoz1 gene 5 's end upstream sequence can interact with the conidial nucleoprotein of Arthobotrys oligospora, make
With rear so that apparent hysteresis occur in DNA bands, and with the increase of nuclear protein concentrations, DNA hysteresis gradually increases,
Show that Aoz1 gene promoter sequences have with the conidial nucleoprotein of Arthobotrys oligospora and combines activity.
4, the structure of the recombination Arthobotrys oligospora of Aoz1 genes double-promoter.
1. the series connection of Aoz1 gene double-promoters.
According to Arthobotrys oligospora Aoz1 gene promoter sequences, i.e.,<210>1, design primer FP1-RP1 and FP2-RP2.
Using recombinant plasmid pT-PAoz1 as template, it is utilized respectively primers F P1-RP1 and FP2-RP2 and is expanded.
Reaction condition is as follows：95 DEG C of pre-degeneration 3min；95 DEG C denaturation 40s, 52 DEG C annealing 40s, 72 DEG C extension 2min, 38
Cycle；72 DEG C of extension l0min.Gel extraction after 1% concentration agarose gel electrophoresis.Then by SOE-PCR technological incorporation,
Downstream homology arm first uses Taq archaeal dna polymerases to extend upstream and downstream homology arm complementation, using 25 μ L reaction systems：Upstream and downstream
Each 2.5 μ L, 2.5mmol/L dNTPs of 3 μ L, 10 × PCR buffer, 1.5 μ L of homology arm, 14.8 μ L, Taq archaeal dna polymerases of water
0.2μL。
Reaction condition：95 DEG C of pre-degeneration 4min；95 DEG C of denaturation 50s, 48 DEG C of annealing 30s, 72 DEG C of extension 3min, 10 are followed
Ring.Each 0.5 μ L of FP1-RP2 primers are added, Aoz1 gene double-promoter PPAoz1 segments are expanded.
Reaction condition：95 DEG C of pre-degeneration 4min；95 DEG C of denaturation 50s, 52 DEG C of annealing 40s, 72 DEG C of extension 2min, 30 are followed
Ring；72 DEG C of extension l0min.Recovery product is connect by gel extraction after 1% concentration agarose gel electrophoresis with pMD18-T carriers,
Structure contains Aoz1 gene double-promoter PPAoz1 segments, obtains recombinant plasmid pT-PPAoz1.
2. recombinating structure and the identification of Arthobotrys oligospora.
With restriction enzyme difference double digestion Agrobacterium tumefaciems (A.tumefaciens) binary vector pUR5750 and again
Group plasmid pT-PPAoz1 is constructed in double-promoter PPAoz1 segments directed cloning to pUR5750 binary vectors
pUR5750-PPAoz1.Hygromycin B resistant gene Hyg is inserted between the left margin of T-DNA and right margin, is marked as screening
Remember gene, the binary vector pUR5750-PPAoz1 electrotransformation methods built are then imported Agrobacterium tumefaciems
(A.tumefaciens) in.The Agrobacterium tumefaciems engineered strain of the binary vector containing pUR5750-PPAoz1 is inoculated with to containing damp mould
In the MM culture solutions of plain B, 28 DEG C, after 200r/min cultivates 1-2d, thalline were collected by centrifugation, by above-mentioned A.tumefaciens bacterium solutions
It is mixed with isometric Arthobotrys oligospora conidium, mixed liquor is coated on the solid CMA for being covered with nitrocellulose filter
In culture medium, after co-culturing 2-3d at 28 DEG C, filter membrane is transferred to containing cefotaxime (200 μm of ol/L) and is contained
In the CMA solid mediums of hygromycin B (30 μ g/ml), 4-5d is cultivated at a temperature of 25 DEG C, until there are resistant clones, according to
Extension rate calculates transformation efficiency.Resistant clones are transferred in the fresh CMA culture mediums containing hygromycin B and are cultivated, are collected
Mycelia extracts the genomic DNA of resistant clones, and the few spore Arthrobotrys of double-promoter recombination are identified by PCR with primers F P3-RP3
As a result bacterium can expand to obtain the genetic fragment of 4100bp sizes, to confirming to contain Aoz1 gene double startups after the sequencing fragment
Sub- PPAoz1 segments.
5, double-promoter recombination Arthobotrys oligospora Aoz1 gene transcription levels detection.
It is special according to the GenBank sequence design detection Arthobotrys oligospora Aoz1 genes logged in and internal standard gene 18s rDNA
Specific primer extracts the double-promoter after the induction of haemonchus contortus stageⅢlarvae respectively according to the step of TrizoL specifications
Arthobotrys oligospora and the source of parents Arthobotrys oligospora total serum IgE with non-recombinant are recombinated, is tried using SYBR Premix Ex TaqTM
Agent box (TaKaRa) carries out fluorescence quantifying PCR method respectively on 4 instruments of Bio-Rad Chromo with primers F P4-RP4
(qRT-PCR) it detects.Each sample is repeated 3 times.The opposite of 2 kinds of strains A oz1 genes is calculated separately according to the method for 2- △ △ CT
Expression quantity carries out difference analysis.The result shows that compared with source of parents Arthobotrys oligospora, the few spore Arthrobotrys of double-promoter recombination
The Aoz1 gene relative transcript levels of bacterium have raised 1.8 times, it was demonstrated that the double-promoter PPAoz1 of structure can significantly improve Aoz1 bases
Because of transcriptional level.
6, double-promoter recombination Arthobotrys oligospora genetic stability analysis.
The recombination Arthobotrys oligospora continuous passage in vitro of Aoz1 gene double-promoters will be built, with primers F P3-RP3
By PCR method respectively to the 5th, 10,15,20,25,30,35,40,45, the double-promoters of 50 generations recombinate Arthobotrys oligospora
Aoz1 Gene Double promoter sequences are detected, analysis recombination Arthobotrys oligospora bacterial strain genetic stability；With primers F P4-RP4
By fluorescence quantifying PCR method (qRT-PCR) respectively to the 5th, 10,15,20,25,30,35,40,45, the double startups of 50 generations
Son recombination Arthobotrys oligospora strains A oz1 gene mRNA levels are detected, analysis recombination Arthobotrys oligospora transcriptional level
Stability.The result shows that after continuously passing for 50 generations, the Aoz1 genes double-promoter of double-promoter recombination Arthobotrys oligospora can be stablized
It is present in recombinant bacterium genome (Fig. 5), it was demonstrated that double-promoter, which recombinates Arthobotrys oligospora, has good genetic stability.With
5th generation double-promoter recombination Arthobotrys oligospora is compared, and the 50th generation double-promoter recombinates the Aoz1 genes of Arthobotrys oligospora
There is not apparent decline in mRNA level in-site, it was demonstrated that double-promoter, which recombinates Arthobotrys oligospora strain, has stable transcriptional level.
7, double-promoter recombinates Arthobotrys oligospora nematode-trapping activity analysis.
1. double-promoter recombinates effect of the Arthobotrys oligospora to haemonchus contortus.
The source of parents Arthobotrys oligospora of the recombination Arthobotrys oligospora and non-recombinant of the double-promoter of gene containing Aoz1 is taken respectively
Mycelia block (0.5cm × 0.5cm) contacts dialysis film surface left-hand thread with mycelia face and is put in 25 DEG C behind dialysis membrane center, label
It is cultivated in incubator.It is compared simultaneously with source of parents Arthobotrys oligospora.It is added when mycelia covers the 2/3 of entire dialysis membrane fresh
Haemonchus contortus stageⅢlarvae makes it be uniformly distributed in entire culture medium dialysis film surface, continues in 25 DEG C of incubator
Culture.12h after polypide is added, for 24 hours, 36h, 48h and 72h when take out, recombinated in microscopically observation double-promoter
Arthobotrys oligospora, source of parents Arthobotrys oligospora are to the predation of haemonchus contortus stageⅢlarvae.As a result, it has been found that effect
After for 24 hours, Arthobotrys oligospora is carried out to form predation ring, and part nematode head or tail portion are entangled, with the extension of time, twisted
Haemonchus is fixed by mycelia, and nematode body wall cuticula is degraded, and then haemonchus contortus is infected by mycelia, inside polypide
Mycelia is overgrowed with, polypide interior tissue is by destruction (Fig. 6).
2. double-promoter recombinates Arthobotrys oligospora and preys on active measurement to haemonchus contortus.
The sheep dung containing haemonchus contortus worm's ovum is collected, 2 groups, every group of 10g are divided into after pulverizing, is separately added into double open
Mover recombinates Arthobotrys oligospora and 10 fritter of source of parents Arthobotrys oligospora mycelia block, is uniformly mixed, third group is as blank pair
According to.It is protected from light culture at 25 DEG C, uses improvement Bell's Mans method to collect remaining larva after 15d, and count, is distinguished using following formula
Calculate double-promoter recombination Arthobotrys oligospora and source of parents Arthobotrys oligospora predation activity.Predation activity=(control group is remaining
Larva number-experimental group residue larva number)/control group residue larva number × 100%.The result shows that source of parents Arthobotrys oligospora is caught
Food activity is 87.6%, and the predation activity that double-promoter recombinates Arthobotrys oligospora is 95.2%, it was demonstrated that double-promoter recombination is few
Spore Arthrobotrys bacterial strain is improved the predation activity of nematode.
The invention has the advantages that：With conventional genetic method for transformation (PEG protoplast transformations, lithium acetate transformation, electric shock
Conversion method and restriction enzyme mediated transformation) it compares, ATMT is with acceptor material range is wide, transformation efficiency is high, transformant stablizes heredity
The advantages that, the bottleneck of Conventional reformat method is overcome, one of the important method of filamentous fungus strain molecular improvement is become.With it is non-heavy
The source of parents Arthobotrys oligospora strain of group is compared, and Arthobotrys oligospora is recombinated using the Aoz1 gene double-promoters of ATMT technologies structure
There is good genetic stability, the enhancing of Aoz1 gene transcription levels, nematode-trapping activity to improve for strain, be ruminant digestion
The research and development of the anti-controlling agent of road nematodiasis biology provide an efficient engineering strain.
Description of the drawings
Fig. 1 is the culture and morphologic observation of nematode-trapping fungi-Arthobotrys oligospora.
In Fig. 1, A-D:The form of Arthobotrys oligospora different incubation time mycelia in CAM culture mediums.
Fig. 2 is the conidial preparation and purification of Arthobotrys oligospora.
In Fig. 2, A：The conidium of formation；B：The conidium of purifying.
Fig. 3 is the amplification of Arthobotrys oligospora Aoz1 gene promoter PAoz1 genetic fragments.
In Fig. 3,1-3：The amplification of Arthobotrys oligospora Aoz1 gene promoters；M：DNA marker DL-2000.
Fig. 4 Arthobotrys oligospora Aoz1 gene promoter PAoz1 transcriptional activities detect.
In Fig. 4：A：Transfect the conidium of empty carrier pPIC9K-EGFP；B：Promoter vector pPIC9K- after transfection
The conidium of PAoz1-EGFP.
Fig. 5 is that double-promoter PPAoz1 recombinates the analysis of Arthobotrys oligospora genetic stability.
In Fig. 5：1-10：PCR method pair the 5th, 10,15,20,25,30,35,40,45, the double-promoters of 50 generations
PPAoz1 recombinates the detection of Arthobotrys oligospora Aoz1 gene promoters；M：DNA marker DL-2000.
Predation activity of Fig. 6 double-promoters PPAoz1 recombination Arthobotrys oligosporas to haemonchus contortus.
In Fig. 6, A：Double-promoter recombinates Arthobotrys oligospora mycelia and forms three dimensional network trapping organs；B：Double-promoter recombinates
Arthobotrys oligospora collarium entangles haemonchus contortus；C：Haemonchus contortus is fixed by mycelia；D：Haemonchus contortus quilt
Mycelia infects the resolution stage.
Fig. 7 is fusion DNA vaccine (SOE-PCR) specific primer table used in 1 present invention of table.
Specific implementation mode
The present invention is described in further detail below by specific embodiment.
1, the culture of Arthobotrys oligospora.
Arthobotrys oligospora strains isolated from Xinjiang XA3 (A.oligospora XA3) conidium deposited of going bail for is inoculated in
On 0.4g/mL corn meal agars culture medium (CMA), (Fig. 1) is cultivated in 25 DEG C of incubators.Cut the mycelia block 5~10 of above-mentioned culture
Fritter is put into the niblet triangular flask of sterilizing, 25 DEG C of culture 21d, with aseptic distillation water elution conidium (Fig. 2), after counting
4 DEG C of refrigerators save backup.
2, the clone of Arthobotrys oligospora Aoz1 gene 5 's end upstream sequence and sequence signature analysis.
1. the design of primers of Arthobotrys oligospora GenomeWalker.
The Universial GenomeWalker produced according to BD companiesTMAdapter-primer sequence in 2.0 kits
AP1：5 '-GTAATACGACTCACTATAGGGC-3' and AP2：5’-ACTATAGGGCACGCGTGGT-3'；With Aoz1 genes
It is template that cDNA 5 ', which holds end sequence, designs the specific primer of its GenomeWalker：GSP1：5 ' ends-
CGGATTGGGCCAGCAAAGGCATTGGTAGC-3'；GSP2：5 ' end-GATTGCTAGAAGGGAGATGAGGCCGTTCG-3'
(table 1).
2. genomic DNA digestion and purifying.
Arthobotrys oligospora conidium is taken, is ground in the mortar that liquid nitrogen is added, the fungi produced with Promega companies
DNA extraction kit extracts Arthobotrys oligospora conidium DNA.With 4 restriction enzyme DraI, EcoRV, PvuII,
StuI carries out digestion to Arthobotrys oligospora DNA respectively.Simultaneously with the genome of the mankind provided in PvuII cleavage reagent boxes
DNA, as positive control.With DNA Purification Kit digestion products, DNA is dissolved in 20 μ L TE solution.
3. the connection of genomic DNA fragment and connector.
Learn from else's experience four kinds of endonuclease digestions and Arthobotrys oligospora genomic DNA fragment after purification and positive control-mankind
DNA-PvuII digestion fragments are connected with GenomeWalker connectors (Adaptor) respectively.
4. Arthobotrys oligospora Aoz1 gene 5 's end upstream sequence PCR amplification.
According to the Universial GenomeWalker produced according to BD companiesTMThe operation of 2.0 kit specifications walks
It is rapid to carry out twice PCR reaction：Initial p CR (primary PCR) and secondary PCR (secondary PCR).Initial p CR is with few spore
Arthrobotrys bacterium genomic DNA fragment is template, is expanded using AP1-GSP1 primers, amplified production size is about 2100bp；
Second of PCR is expanded using initial PCR amplification product as template using AP2-GSP2 primers, and amplified production size is about
2050bp.Each PCR reactions all set negative control and positive control.PCR system is detected with the positive control that kit provides
Feasibility.As a result it has been obtained on the Arthobotrys oligospora Aoz1 gene 5 's end that size is about 2050bp by second of PCR amplification
Swim genetic fragment (Fig. 3).
5. the bioinformatic analysis of Aoz1 gene 5 's end upstream sequence.
After purification by PCR product, pMD-18T carriers are cloned into, construction recombination plasmid pT-PAoz1 send TaKaRa biologies public
Department is sequenced, and the sequence of measurement is carried out sequence alignment and analysis with Blast softwares on GenBank, is opened with eukaryocyte
Mover analysis software (Promoter Scan, Promoter Finder and Promoter Prediction Server) analysis should
The features such as sequence cis-regulating element [TATA-box, GC-box, transcription initiation site Initiator, BRE (TFIIB
Recognition Element)、DPE(Downstream core Promoter Element)：The conservative member of downstream promoter
Part, UPE (Upstream Promoter Element)].As a result clone has obtained the Aoz1 gene 5 's end upstream sequence of 2041bp
Row；Bioinformatic analysis is found, finds that there are TATA box, CAAT box, GC in the upstream sequence of Aoz1 gene 5 's end
Tubby, ZnCys_Ab0079, NF-X1_Pbl0001, Sp1, GCF, PEBP2, EIIF, element_ are also contained in the islands box and CpG
The binding site of the transcription factors such as II_rs-4, E2F, EivF/CREB.
3, Arthobotrys oligospora Aoz1 gene promoter transcriptions Activity determination.
1. the structure of the promoter expression vector containing EGFP reporter genes.
By pPIC9K-EGFP promoter detections carrier SacI and BamHI double digestions, cuts off 5 ' AOX1 on carrier and start
Son recycles carrier large fragment.By recombinant plasmid pT-PAoz1 SacI and BamHI double digestions, gel extraction Aoz1 bases after electrophoresis
Because of promoter PAoz1 segments, by the Aoz1 gene promoter PAoz1 segments of recycling and pPIC9K-EGFP promoter detection carriers
Segment overnight, is converted 100 μ L Top10 competent cells, is selected and obtained using bacterium solution PCR method with the 16 DEG C of connections of T4DNA ligases
Obtain recombinant promoter Expression vector pPIC9K-PAoz1-EGFP positive colonies.
2. recombinant vector converts.
The aseptic double-distilled water that the Arthobotrys oligospora conidium of preparation is pre-chilled is resuspended, then molten with the sorbierite of precooling
Liquid washs conidium, and finally bacterial sediment is resuspended with sorbitol solution, obtains Arthobotrys oligospora conidium competence
Cell.By the Arthobotrys oligospora Aoz1 gene promoter report carriers of pPIC9K-EGFP (empty vectors) and structure
(pPIC9K-PAoz1-EGFP) electrotransformation Arthobotrys oligospora conidium is distinguished under conditions of 1.5kv, 20 μ F and 200 Ω.
After electricity turns, it is added the culture medium of precooling in the cup that shocks by electricity, and by conidium inoculation method corn meal agar culture medium flat plate
Upper culture.
3. the transcription and translation level of reporter gene EGFP detects.
Take respectively pPIC9K-EGFP (blank control group) and with the conidiums of pPIC9K-Aoz1-EGFP processing groups from
The heart after being resuspended with fixer, stands 1h at room temperature.Dyeing liquor is added to dye 4-6h at 37 DEG C, 75% ethanol decolorization is marked with DIG
The EGFP gene nucleic acid probe of note carries out northern blot detections.It as a result can be with the EGFP gene nucleic acid probe of DIG labels
Detect the transcription (mRNA) of EGFP gene；The fluorescence that yellow green is sent out with conidium can be detected with fluorescence microscope
(Fig. 4).
4. Aoz1 gene promoter DNA gel retardation experiments.
According to the Aoz1 gene 5 's end upstream sequence design probe (DePAoz1) of acquisition：5 ' ends-
GGGATACGTACGTGCTTAACTCCGCAATCCGGATGGGATCTTG-3', synthesising probing needle, is produced with Roche companies in vitro
Gel blocking kit (DIG Gel Shift Kit) 5 ' end of probe DePAoz1 sequences is marked, required for acquisition
Nucleic acid probe, measure DNA concentration and purity with ultraviolet specrophotometer, the Nuclear extract for then using Sigma companies to produce
Extracts kit extracts conidial nucleoprotein.By the probe and the careful mixing of nucleoprotein of DIG labels, 15-25 DEG C of placement
15min；5uL sample loading buffers are added, carry out polyacrylamide gel electrophoresis, electrophoresis to blue bands is at bottom 2/3.It will be with
The nylon film filter paper of gel size, which is placed in 0.5 × TBE, balances 5min, carries out transferring film and fixation.By nylon membrane (DNA is face-up)
It is placed in hybridization bag, adds 1mL CSPD working solutions, close hybridization bag, 37 DEG C of incubation 30min.X-ray film is exposed the film to, is carried out
Chemiluminescence detection.As a result Aoz1 gene 5 's end upstream sequence can be with the conidial nucleoprotein phase interaction of Arthobotrys oligospora
With, so that apparent hysteresis occur in DNA bands after effect, and with the increase of nuclear protein concentrations, DNA hysteresis by
It is cumulative strong, show that Aoz1 gene promoter sequences have with the conidial nucleoprotein of Arthobotrys oligospora and combines activity.
4, the structure of the recombination Arthobotrys oligospora of Aoz1 genes double-promoter and identification.
1. the series connection of Aoz1 gene double-promoters.
According to Arthobotrys oligospora Aoz1 gene promoter sequences, design primer FP1-RP1 and FP2-RP2.To recombinate matter
Grain pT-PAoz1 is template, is utilized respectively primers F P1-RP1 and FP2-RP2 and is expanded.Reaction condition is as follows：95 DEG C of pre-degenerations
3min；95 DEG C of denaturation 40s, 52 DEG C of annealing 40s, 72 DEG C of extension 2min, 38 recycle；72 DEG C of extension l0min.1% concentration agar
Gel extraction after sugared gel electrophoresis.Then by SOE-PCR technological incorporation upstream and downstream homology arm, Taq archaeal dna polymerases pair are first used
Upstream and downstream homology arm complementation extends, using 25 μ L reaction systems：Each 2.5 μ of 3 μ L, 10 × PCR buffer of upstream and downstream homology arm
1.5 μ L of L, 2.5mmol/L dNTPs, 14.8 μ L, Taq archaeal dna polymerase of water, 0.2 μ L.Reaction condition：95 DEG C of pre-degeneration 4min；
95 DEG C of denaturation 50s, 48 DEG C of annealing 30s, 72 DEG C of extension 3min, 10 recycle.Each 0.5 μ L of FP1-RP2 primers are added, are expanded
Aoz1 gene double-promoter PPAoz1 segments, reaction condition：95 DEG C of pre-degeneration 4min；95 DEG C of denaturation 50s, 52 DEG C of 40s that anneal, 72
DEG C extend 2min, 30 cycle；72 DEG C of extension l0min.Gel extraction after 1% concentration agarose gel electrophoresis, by recovery product
It is connect with pMD18-T carriers, structure contains Aoz1 gene double-promoter PPAoz1 segments, obtains recombinant plasmid pT-PPAoz1.
2. recombinating structure and the identification of Arthobotrys oligospora.
With restriction enzyme difference double digestion Agrobacterium tumefaciems (A.tumefaciens) binary vector pUR5750 and again
Group plasmid pT-PPAoz1 is constructed in double-promoter PPAoz1 segments directed cloning to pUR5750 binary vectors
pUR5750-PPAoz1.Hygromycin B resistant gene Hyg is inserted between the left margin of T-DNA and right margin, is marked as screening
Remember gene, the binary vector pUR5750-PPAoz1 electrotransformation methods built are then imported Agrobacterium tumefaciems
(A.tumefaciens) in.The Agrobacterium tumefaciems engineered strain of the binary vector containing pUR5750-PPAoz1 is inoculated with to containing damp mould
In the MM culture solutions of plain B, 28 DEG C, after 200r/min cultivates 1-2d, thalline were collected by centrifugation, by above-mentioned A.tumefaciens bacterium solutions
It is mixed with isometric Arthobotrys oligospora conidium, mixed liquor is coated on the solid CMA for being covered with nitrocellulose filter
In culture medium, after co-culturing 2-3d at 28 DEG C, filter membrane is transferred to containing cefotaxime (200 μm of ol/L) and is contained
In the CMA solid mediums of hygromycin B (30 μ g/ml), 4-5d is cultivated at a temperature of 25 DEG C, until there are resistant clones, according to
Extension rate calculates transformation efficiency.Resistant clones are transferred in the fresh CMA culture mediums containing hygromycin B and are cultivated, are collected
Mycelia extracts the genomic DNA of resistant clones, and the few spore Arthrobotrys of double-promoter recombination are identified by PCR with primers F P3-RP3
As a result bacterium can expand to obtain the genetic fragment of 4100bp sizes, to confirming to contain Aoz1 gene double startups after the sequencing fragment
Sub- PPAoz1 segments.
5, double-promoter recombination Arthobotrys oligospora Aoz1 gene transcription levels detection.
It is special according to the GenBank sequence design detection Arthobotrys oligospora Aoz1 genes logged in and internal standard gene 18s rDNA
Specific primer extracts the double-promoter after the induction of haemonchus contortus stageⅢlarvae respectively according to the step of TrizoL specifications
Arthobotrys oligospora and the source of parents Arthobotrys oligospora total serum IgE with non-recombinant are recombinated, is tried using SYBR Premix Ex TaqTM
Agent box (TaKaRa) carries out fluorescence quantifying PCR method respectively on 4 instruments of Bio-Rad Chromo with primers F P4-RP4
(qRT-PCR) it detects.Each sample is repeated 3 times.The opposite of 2 kinds of strains A oz1 genes is calculated separately according to the method for 2- △ △ CT
Expression quantity carries out difference analysis.The result shows that compared with source of parents Arthobotrys oligospora, the few spore Arthrobotrys of double-promoter recombination
The Aoz1 gene relative transcript levels of bacterium have raised 1.8 times, it was demonstrated that the double-promoter PPAoz1 of structure can significantly improve Aoz1 bases
Because of transcriptional level.
6, double-promoter recombination Arthobotrys oligospora genetic stability analysis.
The recombination Arthobotrys oligospora continuous passage in vitro of Aoz1 gene double-promoters will be built, with primers F P3-RP3
By PCR method respectively to the 5th, 10,15,20,25,30,35,40,45, the double-promoters of 50 generations recombinate Arthobotrys oligospora
Aoz1 Gene Double promoter sequences are detected, analysis recombination Arthobotrys oligospora bacterial strain genetic stability；With primers F P4-RP4
By fluorescence quantifying PCR method (qRT-PCR) respectively to the 5th, 10,15,20,25,30,35,40,45, the double startups of 50 generations
Son recombination Arthobotrys oligospora strains A oz1 gene mRNA levels are detected, analysis recombination Arthobotrys oligospora transcriptional level
Stability.The result shows that after continuously passing for 50 generations, the Aoz1 genes double-promoter of double-promoter recombination Arthobotrys oligospora can be stablized
It is present in recombinant bacterium genome (Fig. 5), it was demonstrated that double-promoter, which recombinates Arthobotrys oligospora, has good genetic stability.With
5th generation double-promoter recombination Arthobotrys oligospora is compared, and the 50th generation double-promoter recombinates the Aoz1 genes of Arthobotrys oligospora
There is not apparent decline in mRNA level in-site, it was demonstrated that double-promoter, which recombinates Arthobotrys oligospora strain, has stable transcriptional level.
7, double-promoter recombinates Arthobotrys oligospora nematode-trapping activity analysis.
1. double-promoter recombinates effect of the Arthobotrys oligospora to haemonchus contortus.
The source of parents Arthobotrys oligospora of the recombination Arthobotrys oligospora and non-recombinant of the double-promoter of gene containing Aoz1 is taken respectively
Mycelia block (0.5cm × 0.5cm) contacts dialysis film surface left-hand thread with mycelia face and is put in 25 DEG C behind dialysis membrane center, label
It is cultivated in incubator.It is compared simultaneously with source of parents Arthobotrys oligospora.It is added when mycelia covers the 2/3 of entire dialysis membrane fresh
Haemonchus contortus stageⅢlarvae makes it be uniformly distributed in entire culture medium dialysis film surface, continues in 25 DEG C of incubator
Culture.12h after polypide is added, for 24 hours, 36h, 48h and 72h when take out, recombinated in microscopically observation double-promoter
Arthobotrys oligospora, source of parents Arthobotrys oligospora are to the predation of haemonchus contortus stageⅢlarvae.As a result, it has been found that effect
After for 24 hours, Arthobotrys oligospora is carried out to form predation ring, and part nematode head or tail portion are entangled, with the extension of time, twisted
Haemonchus is fixed by mycelia, and nematode body wall cuticula is degraded, and then haemonchus contortus is infected by mycelia, inside polypide
Mycelia is overgrowed with, polypide interior tissue is by destruction (Fig. 6).
2. double-promoter recombinates Arthobotrys oligospora and preys on active measurement to haemonchus contortus.
The sheep dung containing haemonchus contortus worm's ovum is collected, 2 groups, every group of 10g are divided into after pulverizing, is separately added into double open
Mover recombinates Arthobotrys oligospora and 10 fritter of source of parents Arthobotrys oligospora mycelia block, is uniformly mixed, third group is as blank pair
According to.It is protected from light culture at 25 DEG C, uses improvement Bell's Mans method to collect remaining larva after 15d, and count, is distinguished using following formula
Calculate double-promoter recombination Arthobotrys oligospora and source of parents Arthobotrys oligospora predation activity.Predation activity=(control group is remaining
Larva number-experimental group residue larva number)/control group residue larva number × 100%.The result shows that source of parents Arthobotrys oligospora is caught
Food activity is 87.6%, and the predation activity that double-promoter recombinates Arthobotrys oligospora is 95.2%, it was demonstrated that double-promoter recombination is few
Spore Arthrobotrys bacterial strain is improved the predation activity of nematode.
It the above is only the preferred embodiment of the present invention, it is noted that above-mentioned preferred embodiment is not construed as pair
The limitation of the present invention, protection scope of the present invention should be subject to claim limited range.For the art
For those of ordinary skill, without departing from the spirit and scope of the present invention, can also make several improvements and modifications should also regard
For protection scope of the present invention.
<110>Shihezi Univ
<120>The recombination Arthobotrys oligospora and preparation method thereof of Aoz1 gene double-promoters
<141>
<160> 7
<210> 1
<211> 2041
<212> DNA
<213>Arthobotrys oligospora（Arthrobotrys oligospora）
<220>
<221> misc_feature
<223>Aoz1 gene promoter PAoz1 DNA sequence dnas
<400> 1
-2041 CAGAATGAGG TATCTCCTCA GTCTAGACTA AGCCATGCTT AGCTTTGAAA TTGCCAATCT
-1981 GCACATGAGC CATTATGATC GCGTGATTGA TCCAGCATGA ATATTTAGCT CGTGTGTCAA
-1921 GCTCCGTCAA GAATCCCTAT TCACATCCAC ATCCTTTAAA CTAAATGTCA ACTTGAACCC
-1861 AAGACCGCCC GGCCGTGTTG TGCAATCACA CTTGTGCCAT TTAAGCCAAG AACGATGATC
-1801 ATGCTTCTCC CATGGCTCTC CAATAAACCC AGCTGACTAA CTTCCTAAAT AACCCAAGTT
-1741 CTTGCCGTGT CGTATTAACT TTAGAGTAAT AAGGACGGAT GGCAATCGTC CAGAAATCTC
-1681 CTTCCTGCCT TTTCCAGAAC GACGGGTAGG GCTAGGCACC AAGTCACGCC GTCGGTCGGG
-1621 TTTTCCGAGC CTCGCCTATA CGGCCTTCGA CCTAGTTGTG GCAAAGACGT TCCACATAGA
-1561 TAAGGTTACC AATAGCTATA GAACGGATGA AAACGAATTC CACGACTGGG ATTGGTGACG
-1501 CACATGCCTC GAAGGTGTAC TGAGGTGCGA GATCAACCAA TGTTCCATTG GTAAAGGACG
-1441 GTCCTTTCCA TATTGGCCCT ATATTTCGAG ACCAGCATCC CTCGACGTTG ACACTGCGCT
-1381 GCATCGAGCG GGGTTACGAG CTCAGTCGAC AGTCTTTAGT TTCGCGCGGT CAAAGCGCCC
-1321 CTTGTGATCG GAACACATTT CTGACAGAAT GGTATCATGA AAGACCTTCT CAAAGGGGAA
-1261 TCCATCCTGA TGATATCGCG ACAATCGGTA TTGTACCAAG GCTTCACTCA GATGCCTGTA
-1201 ACAACCATCT CGGAACAAAG AGGCGGGCAC ATCTCAAGAA TTTCCGGTCG GTATATCAAG
-1141 CTTCTAGATT ATAATCTTCG TCCTAAAGAT CAACAGGTAT ATTGCCGAGC CGGGCCATAT
-1081 GCGCAATAGC AGTACGAAAG TGAACAAAGG CTGGAAAGGT ATGTAAGTAC TTATGTACAT
-1021 CAAATTTCGG ATCGGAAGAG GCGTGCATGC GTTTCAAATC TTTCAAAGCT TTCGGGGTAA
-961 TATATATGAG TTGAGCTAAG GTCAGAGGAG CACTCTCGTG GGTCAAAAAC GTTAATTTGA
-901 GGGGGTTTAG CCATCGATAA AGCACAAAAT TGACATTGAC GAGGGATCCC TGTTCGTTTG
-841 TCCGCGCCGA GGCAGCAACA TCTGCAACGT GATATCTAGC GCCGTATAAA CGATATCAGC
-781 TACGAGGATT TCAACATCTC TCAAATAGAA ATAATTAAGA TCTTACGTGG GCAAAATAAC
-721 ACGGCCTTCA TGATTTCTAG ACGTTAGACA GCGGATTAAT GTGTGGCAAA GTTAACGTGA
-661 GACCGGATTA TTTGAATTTG GAGTGGCAAA TGTTCAGCCT TCATGAAGCT CCTCCAGCGC
-601 GAAGGTGTAT GTAGATCGGA ATTCGGTTTC GACCGACAAT TGGCAGATCA AATTCTGTCA
-541 GGAGAATCGC TGTACGGAAG AAATATTACT TTTTCGTATC GTGGAGATAT TCCCTTGGAT
-481 TCATGAAGTT GTGGCTAAAG AATATATGGT ATAGCTTTTC CACGACTAGA TCACCCGAAA
-421 GTAAAACGTC AAGACGTCGA CAACTTCCTA AAACATCAAT AGCTTACAGG CTAAGAATTT
-361 CGACACCATG GTTCTCAGGT TGCAAGGTTA CCATGATACG AACCTTCGAC TGTTATACGA
-301 AGGCCGCTAC ATGCCAAGGT CGTGGATGGT CCACGGGATA CGTACGTGCT TAACTCCGCA
-241 ATCCGGATGG GATCTTGCAC AGTATCCCTA CGATGTTCTC ATCATCGACC TTTGATTAAT
-181 CTAGATGTCG ACAGTAGAGA CATTTTTTCT ATAAATCAAG CTCTTGCGCC CTCGATAGAA
-121 TCAAATCCAC TCCATCATCA GCAGCAGTCT TGAAAGCAAT CATCTTCAGA CCTATAGAGT
-61 TTCATCTTTC CTCCTAGTAT CTCTCGATCT ACAGTACACT TCCATAAATT TCAAATCCAC
-1 A
<210> 2
<211> 29
<212> DNA
<213>Arthobotrys oligospora（Arthrobotrys oligospora）
<220>
<221> misc_feature
<223>Aoz1 gene promoter GenomeWalker primers GSP1
<400> 1
5'- CGGATTGGGCCAGCAAAGGCATTGGTAGC-3'
<210> 3
<211> 29
<212> DNA
<213>Arthobotrys oligospora（Arthrobotrys oligospora）
<220>
<221> misc_feature
<223>Aoz1 gene promoter GenomeWalker primers GSP2
<400> 1
5'-GATTGCTAGAAGGGAGATGAGGCCGTTCG-3
<210> 4
<211> 25
<212> DNA
<213>Arthobotrys oligospora（Arthrobotrys oligospora）
<220>
<221> misc_feature
<223>Aoz1 gene promoter fusion DNA vaccine primers Fs P1
<400> 1
5'-CAGAATGAGGTATCTCCTCAGTCTA -3'
<210> 5
<211> 45
<212> DNA
<213>Arthobotrys oligospora（Arthrobotrys oligospora）
<220>
<221> misc_feature
<223>Aoz1 gene promoter fusion DNA vaccine primers RP1
<400> 1
5'-ACCTGAGGCATTCGACCTCATAGCTTGTGGATTTGAAATTTATGG-3'
<210> 6
<211> 45
<212> DNA
<213>Arthobotrys oligospora（Arthrobotrys oligospora）
<220>
<221> misc_feature
<223>Aoz1 gene promoter fusion DNA vaccine primers Fs P2
<400> 1
5'-ACCTGAGGCATTCGACCTCATAGCTCAGAATGAGGTATCTCCTCA-3'
<210> 7
<211> 25
<212> DNA
<213>Arthobotrys oligospora（Arthrobotrys oligospora）
<220>
<221> misc_feature
<223>Aoz1 gene promoter fusion DNA vaccine primers RP2
<400> 1
5'-TGTGGATTTGAAATTTATGGAAGTG-3'
Claims (2)
1. a kind of preparation method of the recombination Arthobotrys oligospora of Aoz1 genes double-promoter, which is characterized in that it is main comprising with
Lower process
The culture of Arthobotrys oligospora：Existing Arthobotrys oligospora conidium is taken to be inoculated in corn meal agar culture medium,
It is cultivated in 20~30 DEG C of incubators, the mycelia fritter for cutting above-mentioned culture is put into the niblet triangular flask of sterilizing, 20~30 DEG C of trainings
18~25d is supported, with aseptic distillation water elution conidium, Cord blood is spare after counting；
The clone of Arthobotrys oligospora Aoz1 gene 5 's end upstream sequence, including following procedure：
1. the design of primers of Arthobotrys oligospora GenomeWalker：According to the adapter-primer sequence AP1 in existing kit：
5 '-GTAATACGACTCACTATAGGGC-3' and AP2：5’-ACTATAGGGCACGCGTGGT-3'；With the cDNA of Aoz1 genes
5 ' end end sequences are template, design the specific primer of its GenomeWalker：GSP1：5 ' ends-
CGGATTGGGCCAGCAAAGGCATTGGTAGC-3'；GSP2：The ends 5'-GATTGCTAGAAGGGAGATGAGGCCGTTCG-3'；
2. genomic DNA digestion and purifying：The Arthobotrys oligospora conidium after culture is taken, is ground in the mortar that liquid nitrogen is added
Mill, with fungal DNA extraction kits extract Arthobotrys oligospora conidium DNA, with restriction enzyme DraI, EcoRV,
PvuII, StuI carry out digestion to Arthobotrys oligospora DNA respectively, while with the base of the mankind provided in PvuII cleavage reagent boxes
Because of a group DNA, DNA is dissolved in 15~25 μ L TE solution with DNA Purification Kit digestion products as positive control
In；
3. the connection of genomic DNA fragment and connector：It learns from else's experience four kinds of endonuclease digestions and Arthobotrys oligospora gene after purification
Group DNA fragmentation and positive control-human DNA-PvuII digestion fragments, respectively with GenomeWalker connectors (Adaptor) phase
Even；
4. Arthobotrys oligospora Aoz1 gene 5 's end upstream sequence PCR amplification：Carry out twice PCR reaction：Initial p CR (primary
PCR it) is used using Arthobotrys oligospora genomic DNA fragment as template with secondary PCR (secondary PCR), initial p CR
AP1-GSP1 primers are expanded, and second of PCR is expanded using initial PCR amplification product as template using AP2-GSP2 primers
Increase, Arthobotrys oligospora Aoz1 gene 5 's end upstream gene segment is obtained by second of PCR amplification；
5. pMD-18T carriers after purification by PCR product, are cloned into, construction recombination plasmid pT-PAoz1,
As a result clone has obtained the Aoz1 gene 5 's end upstream sequence of 2041bp, i.e.,<210>1,
Arthobotrys oligospora Aoz1 gene promoter transcription Activity determinations, including following procedure：
1. the structure of the promoter expression vector containing EGFP reporter genes：PPIC9K-EGFP promoter detection carriers are used
SacI and BamHI double digestions cut off 5 ' AOX1 promoters on carrier, recycle carrier large fragment, recombinant plasmid pT-PAoz1 is used
SacI and BamHI double digestions, gel extraction Aoz1 gene promoter PAoz1 segments after electrophoresis, by the Aoz1 gene promoters of recycling
Sub- PAoz1 segments are connect overnight for 14~18 DEG C with pPIC9K-EGFP promoter detections carrier segments with T4DNA ligases, conversion
80~120 μ L Top10 competent cells are selected using bacterium solution PCR method and obtain recombinant promoter Expression vector pPIC9K-
PAoz1-EGFP positive colonies；
2. recombinant vector converts：The aseptic double-distilled water that the Arthobotrys oligospora conidium of preparation is pre-chilled is resuspended, then in advance
Cold sorbitol solution washs conidium, and finally bacterial sediment is resuspended with sorbitol solution, obtains Arthobotrys oligospora point
Raw spore competent cell, by the Arthobotrys oligospora Aoz1 gene promoter reports of pPIC9K-EGFP (empty vectors) and structure
Announcement carrier (pPIC9K-PAoz1-EGFP) difference electrotransformation Arthobotrys oligospora conidium after electricity turns, adds in the cup that shocks by electricity
Enter the culture medium of precooling, and will be cultivated on conidium inoculation method corn meal agar culture medium flat plate；
The structure of the recombination Arthobotrys oligospora of Aoz1 gene double-promoters, including following procedure：
1. the series connection of Aoz1 gene double-promoters：According to Arthobotrys oligospora Aoz1 gene promoter sequences, i.e.,<210>1, design
Primers F P1-RP1 and FP2-RP2, using recombinant plasmid pT-PAoz1 as template, be utilized respectively primers F P1-RP1 and FP2-RP2 into
Row amplification, structure contain Aoz1 gene double-promoter PPAoz1 segments, obtain recombinant plasmid pT-PPAoz1,
2. recombinating the structure of Arthobotrys oligospora：Distinguish double digestion Agrobacterium tumefaciems with restriction enzyme
(A.tumefaciens) binary vector pUR5750 and recombinant plasmid pT-PPAoz1, by double-promoter PPAoz1 segments orientation gram
In the grand binary vector to pUR5750, pUR5750-PPAoz1 is constructed, hygromycin B resistant gene Hyg is inserted into T-DNA's
Between left margin and right margin, as riddled basins, then the binary vector pUR5750-PPAoz1 electricity built is turned
Change method imports in Agrobacterium tumefaciems (A.tumefaciens), is inoculated with the crown gall agriculture bar of the binary vector containing pUR5750-PPAoz1
In bacterium engineered strain to the MM culture solutions containing hygromycin B, 26-30 DEG C, after 100-300r/min cultivates 1-2d, bacterium is collected by centrifugation
The A.tumefaciens bacterium solutions are mixed with isometric Arthobotrys oligospora conidium, mixed liquor are coated on paving by body
Have in the solid CMA culture mediums of nitrocellulose filter, after co-culturing 2-3d at 26-30 DEG C, filter membrane is transferred to containing ammonia
In thiophene oxime cephalosporin and CMA solid mediums containing hygromycin B, 4-5d is cultivated at a temperature of 22-28 DEG C, until resisting
Property bacterium colony, calculates transformation efficiency according to extension rate, resistant clones is transferred to the fresh CMA culture mediums containing hygromycin B
Middle culture collects mycelia, extracts the genomic DNA of resistant clones, obtain containing Aoz1 gene double-promoter PPAoz1 segments.
2. the recombination Arthobotrys oligospora and its preparation and preparation method of a kind of Aoz1 genes double-promoter, which is characterized in that root
It is prepared according to method described in claim 1.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113355347A (en) * | 2021-06-09 | 2021-09-07 | 安徽大学 | Method for establishing G418 genetic transformation screening system in Arthrobotrys oligospora |
| CN114774436A (en) * | 2022-03-08 | 2022-07-22 | 云南大学 | Application of AOL-S00006g439 gene in regulation and control of Arthrobotrys oligosporus production catcher |
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- 2018-03-20 CN CN201810227752.XA patent/CN108504679A/en active Pending
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| CN113355347A (en) * | 2021-06-09 | 2021-09-07 | 安徽大学 | Method for establishing G418 genetic transformation screening system in Arthrobotrys oligospora |
| CN114774436A (en) * | 2022-03-08 | 2022-07-22 | 云南大学 | Application of AOL-S00006g439 gene in regulation and control of Arthrobotrys oligosporus production catcher |
| CN114774436B (en) * | 2022-03-08 | 2023-01-13 | 云南大学 | Application of AOL-S00006g439 gene in regulation and control of Arthrobotrys oligosporus production catcher |
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