CN103173358A - Preparation method of pathogenic bacteria protoplast of watermelon fusarium oxysporum - Google Patents
Preparation method of pathogenic bacteria protoplast of watermelon fusarium oxysporum Download PDFInfo
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- CN103173358A CN103173358A CN2011104397011A CN201110439701A CN103173358A CN 103173358 A CN103173358 A CN 103173358A CN 2011104397011 A CN2011104397011 A CN 2011104397011A CN 201110439701 A CN201110439701 A CN 201110439701A CN 103173358 A CN103173358 A CN 103173358A
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Abstract
The invention discloses a preparation method of pathogenic bacteria protoplast of watermelon fusarium oxysporum. The preparation method comprises the following steps of: inoculating conidium cultured in a PSA culture medium on a 150mL PDB culture medium for shake cultivation, then filtering a culture solution, and fully washing filtered hypha through 0.8 mol/L NaCl; and dissolving the washed hypha in lywallzyme liquid, sequentially performing primary centrifugation and secondary centrifugation on the dissolved hypha to obtain spore liquid with concentration of 2-5*107 monomer/mL, and then performing a series of operations on the spore liquid to obtain transformant with stable characters, namely, the pathogenic bacteria protoplast of watermelon fusarium oxysporum, which is applied to green fluorescent protein marking. The preparation method disclosed by the invention has the advantages of laying a foundation for genetic manipulations of watermelon fusarium oxysporum protoplast transformation, genetic expression and the like, greatly shortening research time and improving transformation efficiency.
Description
[technical field]
The present invention relates to a kind of pathogenic bacteria protoplast preparation method, be specifically related to a kind of watermelon blight pathogenic bacteria protoplast preparation method for Green Fluorescent Protein.
[background technology]
Watermelon blight is the most serious disease that causes harm on watermelon due to being infected by Fusarium oxysporum f. sp. niveum (Fusarium oxysporum f.sp.niveum), and greater loss is all caused in each watermelon producing region in the world.Plant in case morbidity namely is difficult to cure, is one of disease that seriously restricts watermelon production.
Green fluorescent protein (Green Fluorescent Protein, GFP) is the reporter gene that has been widely used, and finds in luminescent jellyfish in 1962.Owing to itself being a kind of luminescent protein, have stable, directly perceived, easy to operate photoluminescent property and need not add the external source substrate and just can directly position in viable cell and detect and the advantage such as observation, overcome the deficiency of traditional reporter gene in the molecular biology research.
Green fluorescence protein gene is transformed the watermelon Fusarium oxysporum, and the transformant that obtains inoculation watermelon is studied its infection processs, will provide certain theoretical basis for the control of banana Fusarium oxysporum.
The protoplast transformation method is one of means of transforming of GFP, compares particle bombardment and electrization, and the restriction that it is not used by expensive instrument only needs some conventional instruments of testing laboratory.But protoplast preparation is the key of cytogamy and genetic transformation, and high-quality protoplastis can improve success ratio and the efficient of conversion.Different genus and species, not even with the specialized form bacterial strain because thalline self there are differences, can there are differences in the selection of the cultural method of fresh mycelia and lyase.
[summary of the invention]
Technical problem to be solved by this invention is to provide a kind of watermelon blight pathogenic bacteria protoplast preparation method, for the genetic manipulations such as withered germ of water-melon protoplast transformation, genetic expression lay the foundation, and greatly shortened institute and taken time, improved transformation efficiency.
The present invention solves the problems of the technologies described above by the following technical programs: a kind of watermelon blight pathogenic bacteria protoplast preparation method comprises the steps:
Step 1, the watermelon blight pathogenic bacteria is inoculated on the PSA substratum cultivated 7 days; The conidium that then the PSA culture medium culturing is obtained is inoculated on 150mL PDB substratum; And this PDB substratum is placed in carries out shaking culture 24h on constant-temperature table, Temperature Setting is 28 ℃, rotating speed 180r/min obtains mycelia with nutrient solution through 4 layers of filtered through gauze after shaking culture finishes, and adopts 0.8mol/L NaCl fully to wash the mycelia that obtains;
Step 2, add the mycelia after washing in 10mg/mL lywallzyme enzyme liquid that 5mL prepares in advance, 250mg step 1 in the sterilized triangular flask of 50mL, then this triangular flask is placed in and carries out shaking culture 3-4h on constant-temperature table, Temperature Setting is 30 ℃, rotating speed 80r/min; After finishing, shaking culture adopt 3 layers of lens wiping paper that the solution in triangular flask is filtered, the 15mL conical centrifuge tube that filtrate being placed in of filtering acquisition sterilized carries out rotating speed 4000rpm, 4 ℃ of temperature, when being, 10min's is first centrifugal to collect spore, then abandons supernatant spore is carried out secondary centrifuging with acquisition 2-5 * 10 with changing over to after 1ml STC dissolving again in the 15mL conical centrifuge tube of sterilizing
7The spore liquid of individual/mL, the rotating speed of secondary centrifuging are that 3500rpm, temperature are that normal temperature, time are 10min;
step 3, get 5 μ g plasmid DNA and 200 μ l protoplastiss mixing in centrifuge tube, afterwards with this centrifuge tube standing 20min under room temperature, then add 1-1.25ml 40%PTC to this centrifuge tube, place again gently 20min after mixing to obtain reaction solution under room temperature, then will add in this reaction solution 100mL be cooled to 50 ℃ contain 0.9% agar regeneration culture medium mixing after, fall on the regeneration culture medium that contains 0.9% agar, after cultivating 3-5d, 28 ℃ of inversions obtain transformant, continuously switching 4 times is upper with the stable transformant of screening acquired character to the PDA that contains the 150ug/ml hygromycin B, namely obtain to be used for the watermelon blight pathogenic bacteria protoplastis of Green Fluorescent Protein.
The beneficial effect of a kind of watermelon blight pathogenic bacteria of the present invention protoplast preparation method is: determined the suitable condition that protoplastis forms, for the genetic manipulations such as withered germ of water-melon protoplast transformation, genetic expression lay the foundation, and greatly shortened institute and taken time, improved transformation efficiency.
[embodiment]
In order better a kind of watermelon blight pathogenic bacteria of the present invention protoplast preparation method to be set forth explanation, applied for having exemplified following embodiment.
Embodiment one
1, the preparation in bacterium source
The watermelon blight pathogenic bacteria (137) of identifying is inoculated on the PSA substratum cultivated 7 days, standby.
Two, test reagent
Employing is available from lywallzyme (Lywallzyme) the preparation 10mg/mL lywallzyme enzyme liquid of Guangdong Microbes Inst, particularly, take required lywallzyme enzyme amount, homeo-osmosis agent with 0.8mol/L is dissolved it, and 4 kinds of NaC1, KC1, sucrose (sucrose) and sorbyl alcohols (sorbitol) are selected in this homeo-osmosis agent.
Three, watermelon blight pathogenic bacteria protoplast preparation process
Step 1, will cultivate the conidium that obtained in 7 days in the PSA substratum and be inoculated on the 150mLPDB substratum; And this PDB substratum is placed in carries out shaking culture 24h on constant-temperature table, Temperature Setting is 28 ℃, rotating speed 180r/min obtains mycelia with nutrient solution through 4 layers of filtered through gauze after shaking culture finishes, and adopts 0.8mol/L NaCl fully to wash the mycelia that obtains;
Step 2, add the mycelia after washing in 10mg/mL lywallzyme enzyme liquid that 5mL prepares in advance, 250mg step 1 in the sterilized triangular flask of 50mL, then this triangular flask is placed in that to carry out shaking culture 3.5 Temperature Settings on constant-temperature table be 30 ℃, rotating speed 80r/min; After finishing, shaking culture adopt 3 layers of lens wiping paper that the solution in triangular flask is filtered, the 15mL conical centrifuge tube that filtrate being placed in of filtering acquisition sterilized carries out rotating speed 4000rpm, 4 ℃ of temperature, when being, 10min's is first centrifugal to collect spore, then abandons supernatant spore is carried out secondary centrifuging to obtain 2 * 10 with changing in the 15mL conical centrifuge tube of sterilizing after 1ml STC dissolving again
7The spore liquid of individual/mL, the rotating speed of secondary centrifuging are that 3500rpm, temperature are that normal temperature, time are 10min;
step 3, get 5 μ g plasmid DNA and 200 μ l protoplastiss mixing in centrifuge tube, afterwards with this centrifuge tube standing 20min under room temperature, then add 1ml 40%PTC to this centrifuge tube, place again gently 20min after mixing to obtain reaction solution under room temperature, then will add in this reaction solution 100mL be cooled to 50 ℃ contain 0.9% agar regeneration culture medium mixing after, fall on the regeneration culture medium that contains 0.9% agar, after cultivating 5d, 28 ℃ of inversions obtain transformant, continuously switching 4 times is upper with the stable transformant of screening acquired character to the PDA that contains the 150ug/ml hygromycin B, namely obtain to be used for the watermelon blight pathogenic bacteria protoplastis of Green Fluorescent Protein.
For the purpose of simplifying the description and be convenient to the different place of comparison various embodiments of the present invention, in the explanation of following two other embodiment, only describe for different place, and no longer repeat to give unnecessary details to existing together mutually.
Embodiment two
The present embodiment is with the difference that embodiment one compares: the time of shaking culture is 4h in watermelon blight pathogenic bacteria protoplast preparation process steps two; What in step 2, secondary centrifuging obtained is 3.5 * 10
7The spore liquid of individual/mL; The amount that adds the 40%PTC of centrifuge tube in step 3 is 1.25ml; The time of cultivating on the regeneration culture medium that contains 9% agar in step 3 is 3d.
Embodiment three
The present embodiment is with the difference that embodiment one compares: the time of shaking culture is 3.5h in watermelon blight pathogenic bacteria protoplast preparation process steps two, and what in step 2, secondary centrifuging obtained is 5 * 10
7The spore liquid of individual/mL; The amount that adds the 40%PTC of centrifuge tube in step 3 is 1.13ml; The time of cultivating on the regeneration culture medium that contains 9% agar in step 3 is 4d.
To sum up, the present invention has determined the suitable condition that protoplastis forms, and for the genetic manipulations such as withered germ of water-melon protoplast transformation, genetic expression lay the foundation, and has greatly shortened institute and takes time, and has improved transformation efficiency.
Claims (1)
1. a watermelon blight pathogenic bacteria protoplast preparation method, is characterized in that: comprise the steps:
Step 1, the watermelon blight pathogenic bacteria is inoculated on the PSA substratum cultivated 7 days; The conidium that then the PSA culture medium culturing is obtained is inoculated on 150mL PDB substratum; And this PDB substratum is placed in carries out shaking culture 24h on constant-temperature table, Temperature Setting is 28 ℃, rotating speed 180r/min obtains mycelia with nutrient solution through 4 layers of filtered through gauze after shaking culture finishes, and adopts 0.8mol/L NaCl fully to wash the mycelia that obtains;
Step 2, add the mycelia after washing in 10mg/mL lywallzyme enzyme liquid that 5mL prepares in advance, 250mg step 1 in the sterilized triangular flask of 50mL, then this triangular flask is placed in and carries out shaking culture 3-4h on constant-temperature table, Temperature Setting is 30 ℃, rotating speed 80r/min; After finishing, shaking culture adopt 3 layers of lens wiping paper that the solution in triangular flask is filtered, the 15mL conical centrifuge tube that filtrate being placed in of filtering acquisition sterilized carries out rotating speed 4000rpm, 4 ℃ of temperature, when being, 10min's is first centrifugal to collect spore, then abandon supernatant spore is carried out secondary centrifuging with the spore liquid of acquisition 2-5 * 107/mL with changing over to after 1ml STC dissolving again in the 15mL conical centrifuge tube of sterilize, the rotating speed of secondary centrifuging is that 3500rpm, temperature are that normal temperature, time are 10min;
step 3, get 5 μ g plasmid DNA and 200 μ l protoplastiss mixing in centrifuge tube, afterwards with this centrifuge tube standing 20min under room temperature, then add 1-1.25ml 40%PTC to this centrifuge tube, place again gently 20min after mixing to obtain reaction solution under room temperature, then will add 100mL to be cooled to the 0.9% agar regeneration culture medium that contains of 50 ℃ in this reaction solution, after containing 50ug/mlarmplin and 100ug/ml hygromycin B mixing in this substratum, fall on the regeneration culture medium that contains 0.9% agar, after cultivating 3-5d, 28 ℃ of inversions obtain transformant, continuously switching 4 times is upper with the stable transformant of screening acquired character to the PDA that contains the 150ug/ml hygromycin B, namely obtain to be used for the watermelon blight pathogenic bacteria protoplastis of Green Fluorescent Protein.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103834581A (en) * | 2014-03-21 | 2014-06-04 | 福建农林大学 | Preparation method of green fluorescent protein marked coconut stem bleeding disease bacterium protoplast |
| CN105340545A (en) * | 2015-11-23 | 2016-02-24 | 金陵科技学院 | Seedling stage puncture inoculation method for fusarium oxysporum f. sp niveum resistance |
| CN108728369A (en) * | 2018-05-29 | 2018-11-02 | 中国热带农业科学院环境与植物保护研究所 | Withered germ of water-melon RNAi components FonDCL1 deletion mutants body and its construction method |
| CN110184199A (en) * | 2019-05-23 | 2019-08-30 | 华南农业大学 | A kind of preparation and regeneration method of No. 1 microspecies protoplast of banana blight bacteria |
| CN110923257A (en) * | 2019-11-25 | 2020-03-27 | 山东省科学院生态研究所(山东省科学院中日友好生物技术研究中心) | Nano-clay mediated filamentous fungus genetic transformation method and application thereof |
| CN113684173A (en) * | 2021-09-28 | 2021-11-23 | 中国热带农业科学院热带作物品种资源研究所 | Method for separating watermelon fruit protoplast |
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2011
- 2011-12-23 CN CN2011104397011A patent/CN103173358A/en active Pending
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103834581A (en) * | 2014-03-21 | 2014-06-04 | 福建农林大学 | Preparation method of green fluorescent protein marked coconut stem bleeding disease bacterium protoplast |
| CN103834581B (en) * | 2014-03-21 | 2016-03-30 | 福建农林大学 | The coconut stem of Green Fluorescent Protein rushes down the preparation method of blood germ protoplastis |
| CN105340545A (en) * | 2015-11-23 | 2016-02-24 | 金陵科技学院 | Seedling stage puncture inoculation method for fusarium oxysporum f. sp niveum resistance |
| CN108728369A (en) * | 2018-05-29 | 2018-11-02 | 中国热带农业科学院环境与植物保护研究所 | Withered germ of water-melon RNAi components FonDCL1 deletion mutants body and its construction method |
| CN108728369B (en) * | 2018-05-29 | 2019-09-10 | 中国热带农业科学院环境与植物保护研究所 | Withered germ of water-melon RNAi component FonDCL1 deletion mutant body and its construction method |
| CN110184199A (en) * | 2019-05-23 | 2019-08-30 | 华南农业大学 | A kind of preparation and regeneration method of No. 1 microspecies protoplast of banana blight bacteria |
| CN110184199B (en) * | 2019-05-23 | 2021-04-23 | 华南农业大学 | Preparation and regeneration method of banana fusarium wilt bacterium No. 1 microspecies protoplast |
| CN110923257A (en) * | 2019-11-25 | 2020-03-27 | 山东省科学院生态研究所(山东省科学院中日友好生物技术研究中心) | Nano-clay mediated filamentous fungus genetic transformation method and application thereof |
| CN110923257B (en) * | 2019-11-25 | 2022-08-26 | 山东省科学院生态研究所(山东省科学院中日友好生物技术研究中心) | Nano-clay mediated filamentous fungus genetic transformation method and application thereof |
| CN113684173A (en) * | 2021-09-28 | 2021-11-23 | 中国热带农业科学院热带作物品种资源研究所 | Method for separating watermelon fruit protoplast |
| CN113684173B (en) * | 2021-09-28 | 2023-07-28 | 中国热带农业科学院热带作物品种资源研究所 | Method for separating protoplast of watermelon fruit |
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Application publication date: 20130626 |