CN1807580B - Method for separating and purifying insect pathogenic fungoid thallus from infected insect haemolymph - Google Patents
Method for separating and purifying insect pathogenic fungoid thallus from infected insect haemolymph Download PDFInfo
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Abstract
This invention discloses a method for segregation purifying insect pathogenic fungi thalline from suscepted insect bloodlympha. It is prepared through steps as follows: deal with blood cell of suscepted insects with a series of enzymes to destroys and removes their germ plasms; finally seperate and purify to get the products. Adopt the achieved pathogenic fungi thalline to extract DNA and mRNA, which has no insect germ plasm pollution according to PCR and RT-PCR verification. The DNA and mRNA extracted can be used to construct gene library, it also lays solid foundation for further uncovering entomogenous fungi invading mechanism.
Description
Technical field
The present invention relates to a kind of from susceptible insect hemolymph the method for separation and purification insect pathogenic fungus thalline.
Background technology
Present research for the insect pathogenic fungus Disease-causing gene, it mainly is the abduction delivering sequence label storehouse (to call the EST storehouse in the following text) that is based upon under the different isolated conditions, as under different isolated conditions, inducing the EST storehouse by setting up Metarhizium anisopliae (wide spectrum bacterial strain), analyze a large amount of est sequences, analyze pathogenic fungi back expression of gene situation etc. in invading the host insect body in the hope of utilizing the est sequence of inducing under a large amount of different isolated conditions, but this method was not only consuming time, but also effort, and was difficult to detect the pathogenic fungi gene of specifically expressing under condition of living body.
And the pathogenic fungi thalline that separation and purification is polluted to no host DNA and RNA in the host insect hemolymph after pathogenic fungi infects is the gordian technique of separating with crohn fungal pathogens expressing gene in pin main body.Directly obtain the pathogenic fungi thalline in the host insect body, to separate and to clone the insect pathogenic fungus thalline and invade the gene that express the back in the insect body, study their effects in pathogenesis, be the problem that the insider thirsts for solving always.
Summary of the invention
The object of the present invention is to provide a kind of from susceptible insect hemolymph the direct method of separation and purification insect pathogenic fungus thalline.
The object of the present invention is achieved like this: a kind of from susceptible insect hemolymph the method for separation and purification insect pathogenic fungus thalline, it is characterized in that: adopt enzyme processing carrying out cracking and DNA and RNA thereof to adopt the enzyme processing to degrade the hemocyte of susceptible insect, precipitation separation in addition again, purifying makes the pathogenic fungi thalline; In whole enzymolysis process, adopt polymerase chain reaction (to call PCR in the following text) to detect the conserved sequence monitoring, adopt PCR and reverse transcription-polymerase chain reaction (to call RT-PCR in the following text) to detect the conserved sequence method at last the insect pathogenic fungus thalline is identified.
The hemocyte of above-mentioned susceptible insect is after ultrapure water processing, centrifugation, and its precipitation is carried out scission reaction with Proteinase K; After scission reaction was finished, its centrifugation and nuclease carried out DeR, with its RNA and the DNA of degrading, and final isolated insect pathogenic fungus thalline and the cell debris of being precipitated as; In whole enzymolysis process, whether the conservative gene sequences Design insect Auele Specific Primer of employing insect detects insect nucleic acid and exists, the Auele Specific Primer of insect pathogenic fungus conservative gene sequences Design insect pathogenic fungus has or not the existence of insect pathogenic fungus nucleic acid as detection, conservative gene sequence amount by detecting insect behind PCR and the RT-PCR augmentation detection enzymolysis what and have or not and judge that enzyme cuts process, realize whole enzyme is cut the whole process supervision of process and the insect pathogenic bacteria thalline is identified.
Above-mentioned susceptible insect is meant Asiatic migrotory locust, and above-mentioned pathogenic fungi is meant green muscardine fungus; Add the ultrapure water washing in the hemocyte of above-mentioned susceptible Asiatic migrotory locust, blood cell count: the volume of ultrapure water=3.0 * 10
6Individual: 1000-2000 μ l; Add cell lysis buffer solution, blood cell count in the hemocyte of wash, supernatant liquor being removed in centrifugation: the volume of cell lysis buffer solution=1.0 * 10
6Individual: 100-200 μ l, adding 10% sodium laurylsulfonate (SDS) to final concentration behind the mixing is 1%, add Proteinase K behind the mixing after, 30 ℃ of water-baths 1～2 hour, centrifugation abandoning supernatant, washing precipitation; In precipitation, add the nucleolysis damping fluid again, its add-on in the front the blood cell count of susceptible Asiatic migrotory locust add nucleolysis damping fluid, blood cell count: the volume of nucleolysis damping fluid=1.0 * 10
6Individual: 100-200 μ l, add deoxyribonuclease DNAase I, rnase RNAase A again, make its final concentration be 0.5mg/ml, behind the mixing 30 ℃ water-bath 30-45 minute, centrifugation abandoning supernatant, washing precipitation; Supernatant liquor after each washing is got 10 μ l and is carried out the PCR reaction as template, and 2% agarose gel electrophoresis is with the situation of the degraded of the cracking of analyzing the Asiatic migrotory locust hemocyte and DNA, RNA; Extract DNA or mRNA from precipitation, and be PCR and RT-PCR with Asiatic migrotory locust Auele Specific Primer and green muscardine fungus Auele Specific Primer, whether genetic material and the precipitation of whether having polluted Asiatic migrotory locust with checking are the green muscardine fungus thalline.
Locust conservative gene of the present invention is the histone gene of locust, and the primer that designs with its conservative section is
H1：5-’ACCAAGGCGGCGAGGAAGA-3’；
H2：5’-TCGAAGAGGCCGACGAGGTAG-3’
Pathogenic fungi thalline conservative gene sequence of the present invention is a green muscardine fungus tubulin gene, and the primer that designs with its conservative section is
T3：5’-AGCCGACCATGAAGAAGTGC-3’，
T4：5’-TGCCTCCAGGGTTTCCAGAT-3’
In certain monitor procedure of the present invention, also available other conserved sequences are as supervisory sequence, but it should be noted that, in selected these two sections sequences, can not contain similar fragment, and can design more special primer according to these two sections sequences, in sample, can only amplify the single purpose band for examination.
Above-mentioned cell lysis buffer solution is by the 0.606g Tutofusin tris, 0.584g sodium-chlor, and the 0.102g magnesium chloride hexahydrate adds the 80ml deionized water dissolving, with salt acid for adjusting pH value to 8.0, adds water and is settled to the 100ml composition; Above-mentioned nucleolysis damping fluid is by the 0.606g Tutofusin tris, 0.058g sodium-chlor, and the 0.584g magnesium chloride hexahydrate adds the 80ml deionized water dissolving, with salt acid for adjusting pH value to 7.5, adds water and is settled to the 100ml composition.
The present invention adopts a series of enzymes to handle the hemocyte of susceptible insect, and destroying and to remove the genetic material of insect, and in addition separation and purification makes the insect pathogenic fungus thalline.The insect pathogenic fungus that obtains is used for extracting DNA and mRNA, and through the checking of PCR and RT-PCR, the pollution of no insect genetic material can be used for making up gene library, also takes a firm foundation for further opening the entomogenous fungi infection mechanism.
In a word, the method for the invention is directly to obtain the pathogenic fungi thalline in the host insect body, and to separate and to clone the insect pathogenic fungus thalline and invade the gene that express the back in the locust body, this has great significance to studying their effects in pathogenesis.And the present invention further optimizes the condition of insect blood cell cracking and DNA and RNA degraded, under optimal conditions, hemocyte and DNA and RNA in the susceptible insect hemolymph have not only been removed, and successfully separate and purifying the insect pathogenic fungus thalline, for follow-up study insect pathogenic bacteria thalline is laid a good foundation in gene and their effects in pathogenesis of insect expression in vivo.
Description of drawings
Fig. 1: separation and purification insect pathogenic bacteria thalline schematic flow sheet from susceptible insect hemolymph;
Fig. 2: designed locust Auele Specific Primer and the sensitivity analysis of green muscardine fungus Auele Specific Primer;
Fig. 3: cut back microscopy figure with enzyme before infecting locust hemolymph enzyme and cutting;
Fig. 4: with the locust Auele Specific Primer and the green muscardine fungus Auele Specific Primer result that carries out PCR and RT-PCR of design.
Wherein, Fig. 2-A is locust Auele Specific Primer PCR sensitivity analysis figure as a result, and 1: negative control, 2-6: locust dna profiling amount is respectively 100pg, 10pg, 1pg, 0.1pg, M:MARKER standard base (SB) radix.
Fig. 2-B is green muscardine fungus Auele Specific Primer PCR sensitivity analysis figure as a result, and 1: negative control, 2-5: locust dna profiling amount is respectively 100pg, 10pg, 1pg, M:MARKER standard base (SB) radix.
Fig. 3-A is susceptible locust hemolymph microscopy figure before the enzymolysis, 1: locust hemocyte, 2: green muscardine fungus.
Fig. 3-B is susceptible locust hemolymph microscopy figure behind the enzymolysis, 1: green muscardine fungus.
Fig. 4-A is the green muscardine fungus DNA purity that PCR detects purifying from susceptible insect; 1: negative control: no dna profiling+locust Auele Specific Primer, 2: green muscardine fungus DNA+ locust Auele Specific Primer, 3:10ng green muscardine fungus DNA+0.1pg locust DNA+ locust Auele Specific Primer, 4: locust DNA+ locust Auele Specific Primer, 5: negative control: no dna profiling+green muscardine fungus Auele Specific Primer, 6: positive control: green muscardine fungus genomic dna template+green muscardine fungus Auele Specific Primer, 7: the green muscardine fungus genomic dna template+green muscardine fungus Auele Specific Primer of extraction, 8: the green muscardine fungus genomic dna template+locust Auele Specific Primer of extraction, M:MARKER standard base (SB) radix.
Whether Fig. 4-B is for existing locust mRNA in the green muscardine fungus thallus DNA that carries out the RT-PCR Detection and Extraction with the single-minded primer of locust; 1: negative control: no dna profiling, 2: positive control: 0.1pg locust mRNA, green muscardine fungus genomic dna template+0.1pg locust mRNA that 3:10ng extracts, the green muscardine fungus genomic dna that 4:10ng extracts, M:MARKER standard base (SB) radix.
Embodiment
A kind of from susceptible insect hemolymph the method for separation and purification insect pathogenic fungus thalline, it is characterized in that: adopt enzyme processing carrying out cracking and DNA and RNA thereof to adopt the enzyme processing to degrade the hemocyte of susceptible insect, precipitation separation in addition again, purifying makes the pathogenic fungi thalline; In whole enzymolysis process, adopt PCR to detect the conserved sequence monitoring, adopt PCR and RT-PCR to detect the conserved sequence method at last the insect pathogenic fungus thalline is identified.
In a word, theoretical foundation of the present invention utilizes zooblast and fungal cell structural different and design, because the zooblast outermost layer is by the cytolemma parcel, and cytolemma is to be made of double-deck phosphatide and membranin.So utilize the characteristic deterioration membranin of the special degrade proteins of Proteinase K to reach the purpose of destroying zooblast, and the fungal cell is outer by cell walls, its main component is a polysaccharide, so Proteinase K can not destroy the fungal cell, thereby reaches isolating purpose.Therefore, obtain pathogenic fungi, all can adopt method of the present invention so long as relate to from the animal hemolymph.In addition, the present invention is in enzymolysis process, and what hydrolysis temperature adopted is 30 ℃, approaching with its parasitic conditions (26-3D ℃), does not prepare and adopt 37 ℃ of the optimum temperutures of Proteinase K, purpose to be based on to the infection mechanism of better understanding fungi later on.Avoid causing fungi generation acknowledgement mechanism some to occur and infect irrelevant genetic expression owing to the reason of temperature.
Be example with separation and purification green muscardine fungus thalline in the susceptible Asiatic migrotory locust hemolymph particularly below, the invention will be further described in conjunction with the accompanying drawings, and also described as epimere, the present invention is not limited only to this.
The activation of Metarhizium anisopliae bacterial strain CQMa102 spore: this routine Metarhizium anisopliae bacterial strain CQMa102 (Metarhizium anisopliae var.acridum CQMa102) is that the separation and Culture purifying obtains in the bamboo locust bombys batryticatus body of falling ill, by University Of Chongqing's genetically engineered center separation and purification, the bacterial strain that China Institute of Micro-biology preserves at bacterial classification preservation center, deposit number CGMCC No.0877, its conidium is stored in 30% glycerine solution, preserves in-80 ℃ of Ultralow Temperature Freezers are medium-term and long-term.The Metarhizium anisopliae bacterial strain CQMa102 of-80 ℃ of preservations is inoculated on the 1/4SDA plate culture medium activates, used substratum is by 10～11g/l glucose, 2.3～2.7g/l peptone, 4.8～5.2g/l yeast extract, 19～21g/l agar is formed, and its pH value is transferred to 6.0, the bacterial strain 7～14d that grows under 26～28 ℃ of dark conditions, until producing spore, in 4 ℃ of refrigerators, preserve standby then.
Gently scrape the spore layer with the inoculation shovel, the spore powder that scrapes is suspended in 0.05% tween 80 of sterilization, with the abundant mixing of miniature vortex oscillator, four layers of lens wiping paper filter, and count preparation 10 with blood counting chamber
8The spore suspension of individual/ml. locust is the hungry 2h of elder generation before processing, contains 5.0 * 10 at pronotum with the microsyringe inoculation every locust of 4 ℃ of freezing 30min. again
4～1.0 * 10
5The Oleum Gossypii semen 5 μ l of individual muscardine spore.Asiatic migrotory locust is in 26 ± 2 ℃, and adopt 12h illumination: the illumination mode of 12h dark is raised.
Locust hemolymph after 4 days is infected in collection, and getting the blood position is inboard leg membrana articulata behind the locust.Earlier get the blood position with 70% alcohol disinfecting, after treating that alcohol is done, sting the inboard membrana articulata of a lower back leg with aseptic syringe needle, draw effusive locust blood fast in the 1.5ml centrifuge tube that fills 300～500 μ l anticoagulation damping fluids (3.8% Trisodium Citrate) (centrifuge tube is positioned on ice) with the micro-sampling rifle then.Diluted locust hemolymph, 4 ℃, the centrifugal 5min of 6000rpm.Supernatant discarded (blood plasma) is collected hemocyte.
Add three (blood cell counts: the volume of washing water=3.0 * 10 of ultrapure water washing according to blood cell count
6Individual: 1000 μ l), centrifugally abandon most supernatant liquor as far as possible, add cell lysis buffer solution (0.606g Tutofusin tris according to blood cell count, 0.584g sodium-chlor, 0.102g magnesium chloride hexahydrate, add the 80ml deionized water dissolving,, add water and be settled to 100ml with salt acid for adjusting pH value to 8.0) (blood cell count: the volume of damping fluid=1.0 * 10
6Individual: 100 μ l), adding 10% dialkyl group sodium sulfonate (SDS) to final concentration behind the mixing is 1%, the vortex mixing, after adding Proteinase K, 30 ℃ of water-baths 1～2 hour, centrifugal abandoning supernatant, precipitation is with physiological saline repeated washing three times, centrifugal supernatant discarded, precipitation still by the front the locust blood cell count add nucleolysis damping fluid (0.606g Tutofusin tris, 0.058g sodium-chlor, 0.584g magnesium chloride hexahydrate adds the 80ml deionized water dissolving, with salt acid for adjusting pH value to 7.5, add water and be settled to 100ml), add deoxyribonuclease (DNAase I), rnase (RNAase A) makes its final concentration be 0.5mg/ml, behind the mixing 30 ℃ water-bath 30-45 minute, centrifugal abandoning supernatant, precipitation physiological saline repeated washing three times.Supernatant liquor after each washing is got 10 μ l and is carried out the PCR reaction as template, and 2% agarose gel electrophoresis is with the cracking of analysis locust hemocyte and the situation of dna degradation.Precipitation is extracted DNA or mRNA, and with the locust Auele Specific Primer that designs:
According to the conservative section design of the histone gene of locust primer
H1：5-’ACCAAGGCGGCGAGGAAGA-3’；
H2：5’-TCGAAGAGGCCGACGAGGTAG-3’
Green muscardine fungus Auele Specific Primer: according to green muscardine fungus tubulin gene order design primer
T3：5’-AGCCGACCATGAAGAAGTGC-3’，
T4：5’-TGCCTCCAGGGTTTCCAGAT-3’。
Be PCR and RT-PCR, whether genetic material and the precipitation of whether having polluted locust with checking are green muscardine fungus.
Whether the present invention detects locust nucleic acid with the histone gene of locust as conserved sequence and exists, and after all enzymolysis are finished, detects the 3rd physiology salt solution with PCR and cleans supernatant liquor, according to the brightness of pcr amplification band, have or not and judge the enzymolysis performance; Pcr amplification detects the locust histone gene of examining after enzymolysis is finished in the supernatant liquor, and during the driftlessness amplified production, enzymolysis finishes; The evaluation of described green muscardine fungus thalline is meant with microscopy to be observed, after extracting DNA or mRNA, by PCR and RT-PCR, detect the nucleic acid that whether has polluted locust with green muscardine fungus thalline tubulin gene order design primer H1 and H2, T3 and T4 have or not the existence of green muscardine fungus thalline nucleic acid.PCR and RT-PCR augmentation detection, no locust histone gene target amplification product when green muscardine fungus thalline tubulin gene has the target amplification product, proves that gained is precipitated as the green muscardine fungus thalline.
The result shows, with the DNA that extracts, carry out the PCR reaction with the locust Auele Specific Primer, PCR product electrophoresis result shows that (Fig. 4-A): the green muscardine fungus Auele Specific Primer can amplify the PCR product and (see Fig. 4-A, Lane7), the existence of green muscardine fungus DNA is described, promptly be separated to the green muscardine fungus cell. get the green muscardine fungus DNA that 10ng extracts, do not amplify the PCR product with the locust Auele Specific Primer and (see Fig. 4-A, Lane2,8), add 0.1pg locust genomic dna again, can amplify the PCR product (see Fig. 4-A, Lane3), prove that the DNA that does not have locust in the green muscardine fungus genomic dna that extracts pollutes. the deposit D NA of extraction is with locust Auele Specific Primer 1,2 carry out the RT-PCR reaction, product electrophoresis result: get the green muscardine fungus genomic dna that 10ng extracts, with locust Auele Specific Primer 1,2 do not amplify the RT-PCR product (sees Fig. 4-B, Lane4), adds 1pg locust mRNA again and can amplify the RT-PCR product and (see Fig. 4-B, Lane3), prove the pollution that does not have locust RNA among the green muscardine fungus DNA of proposing.
Claims (5)
1. the method for a separation and purification insect pathogenic fungus thalline from susceptible insect hemolymph, it is characterized in that: adopt Proteinase K processing carrying out cracking and DNA and RNA thereof to adopt the enzyme processing to degrade the hemocyte of susceptible insect, precipitation separation in addition again, purifying makes the pathogenic fungi thalline; In whole enzymolysis process, adopt polymerase chain reaction (to call PCR in the following text) to detect the conserved sequence monitoring, adopt PCR and reverse transcription-polymerase chain reaction (to call RT-PCR in the following text) to detect the conserved sequence method at last the insect pathogenic fungus thalline is identified.
2. as claimed in claim 1 from susceptible insect hemolymph the method for separation and purification insect pathogenic fungus thalline, it is characterized in that: the hemocyte of described susceptible insect through ultrapure water handle, after the centrifugation, its precipitation is carried out scission reaction with Proteinase K; After scission reaction was finished, its centrifugation and nuclease carried out DeR, with its RNA and the DNA of degrading, and final isolated insect pathogenic fungus thalline and the cell debris of being precipitated as; In whole enzymolysis process, the conserved sequence of insect is detected insect nucleic acid whether exist; The primer of insect pathogenic fungus conservative gene sequences Design insect pathogenic fungus has or not the existence of insect pathogenic fungus nucleic acid as detection; Conservative gene by detecting insect behind PCR and the RT-PCR augmentation detection enzymolysis what and have or not and judge that enzyme cuts process, realize whole enzyme is cut the whole process supervision of process and the insect pathogenic fungus thalline is identified.
3. as claimed in claim 1 or 2 from susceptible insect hemolymph the method for separation and purification insect pathogenic fungus thalline, it is characterized in that: described susceptible insect is meant Asiatic migrotory locust, and described pathogenic fungi is meant green muscardine fungus; Add the ultrapure water washing in the hemocyte of described susceptible Asiatic migrotory locust, blood cell count: the volume of ultrapure water=3.0 * 10
6Individual: 1000-2000 μ l; Add cell lysis buffer solution, blood cell count in the hemocyte of wash, supernatant liquor being removed in centrifugation: the volume of cell lysis buffer solution=1.0 * 10
6Individual: 100-200 μ l, adding 10% sodium laurylsulfonate (SDS) to final concentration behind the mixing is 1%, add Proteinase K behind the mixing after, 30 ℃ of water-baths 1～2 hour, centrifugation abandoning supernatant, washing precipitation; In precipitation, add nucleolysis damping fluid, its add-on: blood cell count that count the front: the volume of nucleolysis damping fluid=1.0 * 10 again
6Individual: 100-200 μ l, add deoxyribonuclease DNAase I, rnase RNAase A again, make its final concentration be 0.5mg/ml, behind the mixing 30 ℃ water-bath 30-45 minute, centrifugation abandoning supernatant, washing precipitation; Supernatant liquor after each washing, get 10 μ l and carry out the PCR reaction as template, 2% agarose gel electrophoresis, situation with the degraded of the cracking of analyzing the Asiatic migrotory locust hemocyte and DNA, RNA: from precipitation, extract DNA or mRNA, and be PCR and RT-PCR with Asiatic migrotory locust Auele Specific Primer and green muscardine fungus Auele Specific Primer, whether genetic material and the precipitation of whether having polluted Asiatic migrotory locust with checking are the green muscardine fungus thalline.
4. as claimed in claim 3 from susceptible insect hemolymph the method for separation and purification insect pathogenic fungus thalline, it is characterized in that: the locust conservative gene that is adopted is the histone gene of locust, and the primer that designs with its conservative section is
H1：5-’ACCAAGGCGGCGAGGAAGA-3’；
H2：5’-TCGAAGAGGCCGACGAGGTAG-3’
The pathogenic fungi thalline conservative gene sequence that is adopted is a green muscardine fungus tubulin gene, and the primer that designs with its conservative section is
T3：5’-AGCCGACCATGAAGAAGTGC-3’，
T4：5’-TGCCTCCAGGGTTTCCAGAT-3’。
5. as claimed in claim 3 from susceptible insect hemolymph the method for separation and purification insect pathogenic fungus thalline, it is characterized in that: described cell lysis buffer solution is by the 0.606g Tutofusin tris, 0.584g sodium-chlor, 0.102g magnesium chloride hexahydrate, add the 80ml deionized water dissolving, with salt acid for adjusting pH value to 8.0, add water and be settled to the 100ml composition; Above-mentioned nucleolysis damping fluid is by the 0.606g Tutofusin tris, 0.058g sodium-chlor, and the 0.584g magnesium chloride hexahydrate adds the 80ml deionized water dissolving, with salt acid for adjusting pH value to 7.5, adds water and is settled to the 100ml composition.
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| CN102670660B (en) * | 2012-05-30 | 2013-09-25 | 贵州大学 | Processing method and application of aspongopus |
| CN106754406A (en) * | 2016-12-01 | 2017-05-31 | 重庆市中药研究院 | The method and detection liquid-phase chip of aweto are isolated and purified in a kind of body fluid from host of Cordyceps sinensis |
| CN108642193A (en) * | 2018-04-23 | 2018-10-12 | 湖北省农业科学院经济作物研究所 | A kind of separation of entomopathogenic bacterium and identification method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1448502A (en) * | 2002-12-16 | 2003-10-15 | 张泽华 | Green muscardine and use thereof |
| CN1542123A (en) * | 2003-11-07 | 2004-11-03 | 重庆大学 | Metarhizium anisopliae |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1448502A (en) * | 2002-12-16 | 2003-10-15 | 张泽华 | Green muscardine and use thereof |
| CN1542123A (en) * | 2003-11-07 | 2004-11-03 | 重庆大学 | Metarhizium anisopliae |
Non-Patent Citations (2)
| Title |
|---|
| 李菁菁,等.ITS-RFLP分析进行昆虫病原线虫斯氏属和异小杆属的分类鉴定.昆虫天敌24 3.2002,24(3),97-104. |
| 李菁菁,等.ITS-RFLP分析进行昆虫病原线虫斯氏属和异小杆属的分类鉴定.昆虫天敌24 3.2002,24(3),97-104. * |
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