CN103451110A - Method for preparing and regenerating phomopasis asparagi protoplast - Google Patents
Method for preparing and regenerating phomopasis asparagi protoplast Download PDFInfo
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- CN103451110A CN103451110A CN2013103826697A CN201310382669A CN103451110A CN 103451110 A CN103451110 A CN 103451110A CN 2013103826697 A CN2013103826697 A CN 2013103826697A CN 201310382669 A CN201310382669 A CN 201310382669A CN 103451110 A CN103451110 A CN 103451110A
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Abstract
The invention discloses a method for preparing and regenerating a phomopasis asparagi protoplast, which relates to the technical field of fungus protoplast preparation and regeneration in cell engineering. The method comprises the following detailed operation steps of: (1) preparing a phomopasis asparagi conidium suspension; (2) preparing a fresh mycelium; (3) performing enzymolysis on mycelium cell walls; (4) separating the protoplast; and (5) regenerating the protoplast. The method is easy to operate, low in requirements on equipment and short in enzymolysis time and regeneration period, and effectively enhances the efficiency of preparation and regeneration of the protoplast.
Description
Technical field
What the present invention relates to is the preparation of fungi protoplastis and regeneration techniques field in cell engineering, is specifically related to a kind of asparagus stem wilt bacteria protoplastis preparation and renovation process.
Background technology
Asparagus (Asparagus officinalis L.), have another name called officinalis, belongs to Asparagaceae Asparagus perennial root per nnial herb, is a kind of very health vegetables of high nutritive value that have, and enjoys in the world the good reputation of " kings of vegetables ".Asparagus Stem Blight is the worldwide important fungal disease caused by Radix Asparagi Phomopsis (Phmopsis asparagi), in the U.S., Europe and the area such as Australian, report is all arranged.This disease main harm cane or tender bamboo shoot (stems or spears), also can infect the branch stalk and intend leaf, just be water stain shape streak, after crossfade into the bar shaped of furvous shuttle, scab middle part russet depression, a large amount of pore shape pycnidiums are scattered, and it is upper, finally causes extremely adjacent plant of the withered and rapid infection of whole strain.China is as asparagus main producing region, the world, stem wilt generation especially severe, and average infection rate surpasses 50% in recent years, often causes total crop failure in blocks, has become one of main bottleneck of restriction China asparagus development, is known as " asparagus cancer ".At present, utilize protoplastis to carry out the important component part that the molecule transformation technology has become filamentous fungus conversion, associated molecule clone technology and mechanism of causing a disease research, protoplastis molecule transformation technology depends on the efficient reproducible protoplastis of preparation, but due to different sorts filamentous fungal cells structure, especially the structure and composition of cell walls exists notable difference, and the top condition and the system that therefore prepare protoplastis also exist very big-difference.The protoplastis that there is not yet asparagus stem wilt bacteria both at home and abroad prepares the system relevant report, and this is unfavorable for this pathogenic bacteria is carried out to deep pathogenic Study on Molecular Mechanism.The highly efficient regeneration of protoplastis is the requirement of furtheing investigate its pathogenic molecular mechanism the later stage, and different enzymolysis times and steady penetration enhancer have material impact to protoplast regeneration.In order to screen asparagus stem wilt bacteria Variation of Virulence bacterial strain, strengthen the understanding to its mechanism of causing a disease, provide basis for carrying out disease-resistant molecular breeding in agriculture production better, need to be studied the method for its protoplastis preparation and regeneration.
Summary of the invention
For the deficiency existed on prior art, the present invention seeks to be to provide a kind of asparagus stem wilt bacteria protoplastis preparation and renovation process, make the number for preparing of its protoplastis be up to 2.8 * 10
7individual/mL, regeneration rate is up to 24.0%.
To achieve these goals, the present invention realizes by the following technical solutions: a kind of asparagus stem wilt bacteria protoplastis preparation and renovation process, and concrete operation step is as follows:
(1), prepare asparagus stem wilt bacteria conidium suspension:
Picking asparagus stem wilt bacteria Phomopsis asparagi mycelia one ring, be seeded on the solid inclined-plane of being made by 50mL oat nutrient agar, cultivate 10-14d under 25 ℃ of temperature, illumination 12h/d condition, after ripe asparagus stem wilt bacteria pycnidium (can secrete conidium) occurring, add the 30mL sterile distilled water to wash spore, obtain the spore liquid that contains the spore agglomerate, with pipettor, this spore liquid vibration is smashed to the spore agglomerate, filter, get filtrate and carry out doubling dilution, under microscope, count, obtaining concentration is 1 * 10
6the asparagus stem wilt bacteria conidium suspension of individual/mL;
(2), prepare fresh mycelia:
By the conidium suspension inoculation for preparing, in the liquid perfect medium, under 25 ℃ of conditions, the 150r/min shaking table is cultivated 72h, and funnel filters, and obtains fresh mycelia;
(3), enzymolysis hyphal cell wall:
Get the 1.0g fresh mycelia, add the mixed enzyme solution of 10mL lyase, driselase and helicase, 33 ℃ of water enzyme digestion 4.5h, adopt 1.0mol/L MgSO
4(the PBS preparation of 10m mol/L pH6.98, volume ratio is MgSO
4: while PBS=3:1) doing the homeo-osmosis agent, protoplastis quantity reaches 2.8 * 10
7individual/mL;
(4), protoplastis separates:
Filter enzymolysis solution, elimination is not by the mycelia relic of enzymolysis, and filtrate is in 4 ℃ of temperature, rotating speed 5000r/min, centrifugation 5min; Abandon supernatant liquor, with the Sorbitol Solution USP washing of 1-2mL concentration 1.0mol/L 2-3 time, the collection protoplastis is resuspended in the Sorbitol Solution USP of 1mL concentration 1.0mol/L precooling and preserves, and obtains the protoplastis suspension;
(5), protoplast regeneration:
By 100 times of the sucrose solution dilutions of concentration 0.6mol/L for the protoplastis suspension, get diluent 0.1mL and coat the dull and stereotyped and height of potato dextrose agar and ooze the potato dextrose agar flat board; 25 ℃ of temperature, maintenance relative humidity 60%-75%, illumination cultivation 12h/d, cultivate 3-5d until grow diameter 1mm regeneration bacterium colony; Investigation regeneration situation, to the colony count (A) formed; Compare (B) with the PDA substratum that does not contain steady penetration enhancer.Calculate regeneration rate, the total protoplastis number of regeneration rate (%)=(A-B) * 100/;
Described step (3) mixed enzyme solution is comprised of the material of following weight proportion: lyase 15g/L, driselase 10g/L, helicase 15g/L, solvent are 1.0mol/L MgSO
4(the PBS preparation of 10m mol/LpH6.98, volume ratio is MgSO
4: PBS=3:1), configure the rear ultra micro filtering with microporous membrane degerming that is 0.45 μ m with diameter;
Height in described step (5) oozes potato dextrose agar and is comprised of the material of following weight proportion: potato 200g/L, glucose 20g/L, agar 20g/L, sucrose 205g/L.
The invention has the beneficial effects as follows:
1. preparation technology is simple, easy to operate;
2. protoplastis prepares number and is up to 1.5-2.8 * 10
7individual/mL, regeneration rate is up to 24.0%.
Embodiment
For technique means, creation characteristic that the present invention is realized, reach purpose and effect is easy to understand, below in conjunction with embodiment, further set forth the present invention.
This embodiment is by the following technical solutions: a kind of asparagus stem wilt bacteria protoplastis preparation and renovation process, and concrete operation step is as follows:
(1), prepare asparagus stem wilt bacteria conidium suspension:
Picking asparagus stem wilt bacteria Phomopsis asparagi mycelia one ring, be seeded on the solid inclined-plane of being made by 50mL oat nutrient agar, cultivate 10-14d under 25 ℃ of temperature, illumination 12h/d condition, after ripe asparagus stem wilt bacteria pycnidium (can secrete conidium) occurring, add the 30mL sterile distilled water to wash spore, obtain the spore liquid that contains the spore agglomerate, with pipettor, this spore liquid vibration is smashed to the spore agglomerate, filter, get filtrate and carry out doubling dilution, under microscope, count, obtaining concentration is 1 * 10
6the asparagus stem wilt bacteria conidium suspension of individual/mL;
(2), prepare fresh mycelia:
By the conidium suspension inoculation for preparing, in the liquid perfect medium, under 25 ℃ of conditions, the 150r/min shaking table is cultivated 72h, and funnel filters, and obtains fresh mycelia;
(3), enzymolysis hyphal cell wall:
Get the 1.0g fresh mycelia, add the mixed enzyme solution of 10mL lyase, driselase and helicase, 33 ℃ of water enzyme digestion 4.5h, adopt 1.0mol/L MgSO
4(the PBS preparation of 10m mol/L pH6.98, volume ratio is MgSO
4: PBS=3: while 1) doing the homeo-osmosis agent, protoplastis quantity reaches 2.8 * 10
7individual/mL;
(4), protoplastis separates:
Filter enzymolysis solution, elimination is not by the mycelia relic of enzymolysis, and filtrate is in 4 ℃ of temperature, rotating speed 5000r/min, centrifugation 5min; Abandon supernatant liquor, with the Sorbitol Solution USP washing of 1-2mL concentration 1.0mol/L 2-3 time, the collection protoplastis is resuspended in the Sorbitol Solution USP of 1mL concentration 1.0mol/L precooling and preserves, and obtains the protoplastis suspension;
(5), protoplast regeneration:
By 100 times of the sucrose solution dilutions of concentration 0.6mol/L for the protoplastis suspension, get diluent 0.1mL and coat the dull and stereotyped and height of potato dextrose agar and ooze the potato dextrose agar flat board; 25 ℃ of temperature, maintenance relative humidity 60%-75%, illumination cultivation 12h/d, cultivate 3-5d until grow diameter 1mm regeneration bacterium colony; Investigation regeneration situation, to the colony count (A) formed; Compare (B) with the PDA substratum that does not contain steady penetration enhancer.Calculate regeneration rate, the total protoplastis number of regeneration rate (%)=(A-B) * 100/;
Described step (3) mixed enzyme solution is comprised of the material of following weight proportion: lyase 15g/L, driselase 10g/L, helicase 15g/L, solvent are 1.0mol/L MgSO
4(the PBS preparation of 10m mol/LpH6.98, volume ratio is MgSO
4: PBS=3: 1), configure the rear ultra micro filtering with microporous membrane degerming that is 0.45 μ m with diameter;
Height in described step (5) oozes potato dextrose agar and is comprised of the material of following weight proportion: potato 200g/L, glucose 20g/L, agar 20g/L, sucrose 205g/L.
Asparagus stem wilt bacteria protoplastis size homogeneous prepared by the described method of this embodiment, the form sub-circular, it is higher that protoplastis prepares number.Filamentous fungus is due to its structure, and the separation of protoplastis has its unique distinction, and the protoplastis of early origin is from the mycelia tip, and these protoplastiss often lack nucleus, regenerates more difficult; The protoplastis that later stage forms is from the far field cell, the protoplastis heterogeneity of formation.Therefore the present invention adopts the conidium inoculation, fully is grown to the fresh children mycelia in tender period (72h) in conidium and prepares protoplastis, for obtaining higher protoplast yield, provides the foundation.
The protoplast yield of the described method hyphal cell of this embodiment is higher, and the protoplastis vigor of acquisition is also higher.Different lytic enzymes, homeo-osmosis agent etc. are to the formation of the enzymolysis of hyphal cell wall and protoplastis and regenerate extremely important.The present invention selects the MgSO of 1.0mol/L
4(the PBS preparation of 10m mol/L pH6.98, volume ratio is MgSO
4: PBS=3:1) as the homeo-osmosis agent, lytic enzyme is mixed by a certain percentage by lyase, driselase and helicase, for efficiently preparing protoplastis, provides important foundation.The method of the invention adopts the high potato dextrose agar solid medium that oozes of sucrose of 0.6mol/L as regenerated plate, has improved the regeneration rate of protoplastis.Asparagus stem wilt bacteria protoplastis prepared by the method for the invention prepares the high energy of number and reaches 2.8 * 10
7individual/mL, regeneration rate is up to 24.0%.
The method for preparing protoplast such as this embodiment and supersonic method are compared, and the method for the invention easy handling is low for equipment requirements, simultaneously enzymolysis time and regeneration period short, effectively improved the efficiency of protoplastis preparation and regeneration.
It is high that asparagus stem wilt bacteria protoplastis preparation of the present invention and renovation process protoplastis prepare number, regeneration rate is higher, be the reliable method of the preparation of asparagus stem wilt bacteria protoplastis and regeneration, and provide Technical Reference for Phomopsis bacterium protoplastis prepares and regenerates.The Formation and regeneration that the present invention is protoplastis provides a kind of practical significance working method that has, and for further adopting the pathogenic molecular mechanism of protoplast transformation technical study asparagus stem wilt bacteria, provides the foundation.
Example one: the conidium of collecting the pycnidium generation of (OA) on the oat solid substratum, and be inoculated in (CM) in the completely liq substratum, under 25 ℃ of conditions, the 150r/min shaking table is cultivated 36h, obtain fresh mycelia, adopt the mixed enzyme of 1.5% lyase, 1.0% driselase 1.5% helicase: pH6.00, at 30 ℃ of water enzyme digestion 3.0h, use 1.0mol/L MgSO
4(10m mol/LPBS preparation, volume ratio is MgSO
4: PBS=3:1) do the homeo-osmosis agent; By 100 times of the sucrose solution dilutions of concentration 0.6mol/L for the protoplastis suspension, to get diluent 0.1mL and coat the dull and stereotyped and height of potato dextrose agar and ooze the potato dextrose agar flat board cultivate 3-5d under 25 ℃, protoplastis quantity reaches 1.7 * 10
7individual/mL, regeneration rate is 22.0%.
Example two: the conidium of collecting the pycnidium generation of (OA) on the oat solid substratum, and be inoculated in (CM) in the completely liq substratum, under 25 ℃ of conditions, the 150r/min shaking table is cultivated 48h, obtain fresh mycelia, adopt the mixed enzyme of 1.5% lyase, 1.0% driselase 1.5% helicase: pH7.60, at 35 ℃ of water enzyme digestion 4.0h, use 1.0mol/L MgSO
4(10m mol/LPBS preparation, volume ratio is MgSO
4: PBS=3:1) do the homeo-osmosis agent; By 100 times of the sucrose solution dilutions of concentration 0.6mol/L for the protoplastis suspension, to get diluent 0.1mL and coat the dull and stereotyped and height of potato dextrose agar and ooze the potato dextrose agar flat board cultivate 3-5d under 25 ℃, protoplastis quantity reaches 2.2 * 10
7individual/mL, regeneration rate is 22.5%.
Example three: the conidium of collecting the pycnidium generation of (OA) on the oat solid substratum, and be inoculated in (CM) in the completely liq substratum, under 25 ℃ of conditions, the 150r/min shaking table is cultivated 72h, obtain fresh mycelia, adopt the mixed enzyme of 1.5% lyase, 1.0% driselase 1.5% helicase: pH6.98, at 33 ℃ of water enzyme digestion 4.5h, use 1.0mol/L MgSO
4(10m mol/LPBS preparation, volume ratio is MgSO
4: PBS=3:1) do the homeo-osmosis agent; By 100 times of the sucrose solution dilutions of concentration 0.6mol/L for the protoplastis suspension, to get diluent 0.1mL and coat the dull and stereotyped and height of potato dextrose agar and ooze the potato dextrose agar flat board cultivate 3-5d under 25 ℃, protoplastis quantity reaches 2.8 * 10
7individual/mL, regeneration rate is 24.0%.
Above demonstration and described ultimate principle of the present invention and principal character and advantage of the present invention.The technician of the industry should understand; the present invention is not restricted to the described embodiments; that in above-described embodiment and specification sheets, describes just illustrates principle of the present invention; without departing from the spirit and scope of the present invention; the present invention also has various changes and modifications, and these changes and improvements all fall in the claimed scope of the invention.The claimed scope of the present invention is defined by appending claims and equivalent thereof.
Claims (3)
1. prepared and renovation process by an asparagus stem wilt bacteria protoplastis, it is characterized in that, concrete operation step is as follows:
(1), prepare asparagus stem wilt bacteria conidium suspension:
Picking asparagus stem wilt bacteria Phomopsis asparagi mycelia one ring, be seeded on the solid inclined-plane of being made by 50mL oat nutrient agar, cultivate 10-14d under 25 ℃ of temperature, illumination 12h/d condition, after ripe asparagus stem wilt bacteria pycnidium (can secrete conidium) occurring, add the 30mL sterile distilled water to wash spore, obtain the spore liquid that contains the spore agglomerate, with pipettor, this spore liquid vibration is smashed to the spore agglomerate, filter, get filtrate and carry out doubling dilution, under microscope, count, obtaining concentration is 1 * 10
6the asparagus stem wilt bacteria conidium suspension of individual/mL;
(2), prepare fresh mycelia:
By the conidium suspension inoculation for preparing, in the liquid perfect medium, under 25 ℃ of conditions, the 150r/min shaking table is cultivated 72h, and funnel filters, and obtains fresh mycelia;
(3), enzymolysis hyphal cell wall:
Get the 1.0g fresh mycelia, add the mixed enzyme solution of 10mL lyase, driselase and helicase, 33 ℃ of water enzyme digestion 4.5h, adopt 1.0mol/L MgSO
4(the PBS preparation of 10m mol/L pH6.98, volume ratio is MgSO
4: PBS=3: while 1) doing the homeo-osmosis agent, protoplastis quantity reaches 2.8 * 10
7individual/mL;
(4), protoplastis separates:
Filter enzymolysis solution, elimination is not by the mycelia relic of enzymolysis, and filtrate is in 4 ℃ of temperature, rotating speed 5000r/min, centrifugation 5min; Abandon supernatant liquor, with the Sorbitol Solution USP washing of 1-2mL concentration 1.0mo1/L 2-3 time, the collection protoplastis is resuspended in the Sorbitol Solution USP of 1mL concentration 1.0mol/L precooling and preserves, and obtains the protoplastis suspension;
(5), protoplast regeneration:
By 100 times of the sucrose solution dilutions of concentration 0.6mol/L for the protoplastis suspension, get diluent 0.1mL and coat the dull and stereotyped and height of potato dextrose agar and ooze the potato dextrose agar flat board; 25 ℃ of temperature, maintenance relative humidity 60%-75%, illumination cultivation 12h/d, cultivate 3-5d until grow diameter 1mm regeneration bacterium colony; Investigation regeneration situation, to the colony count (A) formed; Compare (B) with the PDA substratum that does not contain steady penetration enhancer.Calculate regeneration rate, the total protoplastis number of regeneration rate (%)=(A-B) * 100/.
2. prepared and renovation process by a kind of asparagus stem wilt bacteria protoplastis according to claim 1, it is characterized in that, described step (3) mixed enzyme solution is comprised of the material of following weight proportion: lyase 15g/L, driselase 10g/L, helicase 15g/L, solvent are 1.0mol/L MgSO
4(the PBS preparation of 10m mol/L pH6.98, volume ratio is MgSO
4: PBS=3:1), configure the rear ultra micro filtering with microporous membrane degerming that is 0.45 μ m with diameter.
3. prepared and renovation process by a kind of asparagus stem wilt bacteria protoplastis according to claim 1, it is characterized in that, height in described step (5) oozes potato dextrose agar and is comprised of the material of following weight proportion: potato 200g/L, glucose 20g/L, agar 20g/L, sucrose 205g/L.
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|---|---|---|---|---|
| CN105176847A (en) * | 2015-11-03 | 2015-12-23 | 中国科学院武汉植物园 | Quick induced spore production method and application of Phomopsis |
| CN105838629A (en) * | 2016-05-16 | 2016-08-10 | 江西省农业科学院蔬菜花卉研究所 | Genetic transformation method for PEG-mediated phomopsis asparagi protoplast |
| CN107058132A (en) * | 2017-05-11 | 2017-08-18 | 中国农业科学院植物保护研究所 | The preparation method of T contraversa protoplast |
| CN107236671A (en) * | 2017-05-11 | 2017-10-10 | 中国农业科学院植物保护研究所 | The preparation method of wheat light Tilletia foetida protoplast |
| CN107422049A (en) * | 2017-04-19 | 2017-12-01 | 江西省农业科学院蔬菜花卉研究所 | A kind of extracting method of asparagus rhizosphere Organic Acids In Soil |
| CN107904178A (en) * | 2017-12-12 | 2018-04-13 | 福建农林大学 | A kind of method for preparing protoplast for not strangling bacterium |
| CN109136113A (en) * | 2018-11-13 | 2019-01-04 | 广西南亚热带农业科学研究所 | A kind of preparation of sugarcane top rot pathogenic bacteria protoplast and regeneration method |
| CN109161480A (en) * | 2018-08-09 | 2019-01-08 | 南京师范大学 | The method for preparing protoplast and gene knockout method of Phomopsis |
| 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 |
| CN111705004A (en) * | 2020-07-24 | 2020-09-25 | 天津市植物保护研究所 | Preparation and regeneration method of biocontrol fungus Acremonium cladosporium protoplast of root-knot nematode |
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- 2013-08-21 CN CN201310382669.7A patent/CN103451110B/en not_active Expired - Fee Related
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| CN105176847B (en) * | 2015-11-03 | 2018-01-09 | 中国科学院武汉植物园 | A kind of rapid induction production spore method and its application of Phomopsis bacterium |
| CN105176847A (en) * | 2015-11-03 | 2015-12-23 | 中国科学院武汉植物园 | Quick induced spore production method and application of Phomopsis |
| CN105838629A (en) * | 2016-05-16 | 2016-08-10 | 江西省农业科学院蔬菜花卉研究所 | Genetic transformation method for PEG-mediated phomopsis asparagi protoplast |
| CN107422049A (en) * | 2017-04-19 | 2017-12-01 | 江西省农业科学院蔬菜花卉研究所 | A kind of extracting method of asparagus rhizosphere Organic Acids In Soil |
| CN107236671B (en) * | 2017-05-11 | 2021-02-26 | 中国农业科学院植物保护研究所 | Preparation method of Tilletia foetida protoplast |
| CN107058132A (en) * | 2017-05-11 | 2017-08-18 | 中国农业科学院植物保护研究所 | The preparation method of T contraversa protoplast |
| CN107236671A (en) * | 2017-05-11 | 2017-10-10 | 中国农业科学院植物保护研究所 | The preparation method of wheat light Tilletia foetida protoplast |
| CN107904178A (en) * | 2017-12-12 | 2018-04-13 | 福建农林大学 | A kind of method for preparing protoplast for not strangling bacterium |
| CN107904178B (en) * | 2017-12-12 | 2021-06-01 | 福建农林大学 | Preparation method of protoplast of basidiomycetes |
| CN109161480A (en) * | 2018-08-09 | 2019-01-08 | 南京师范大学 | The method for preparing protoplast and gene knockout method of Phomopsis |
| CN109161480B (en) * | 2018-08-09 | 2022-01-11 | 南京师范大学 | Preparation method and gene knockout method of protoplast of phomopsis |
| CN109136113A (en) * | 2018-11-13 | 2019-01-04 | 广西南亚热带农业科学研究所 | A kind of preparation of sugarcane top rot pathogenic bacteria protoplast and regeneration 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 |
| CN111705004A (en) * | 2020-07-24 | 2020-09-25 | 天津市植物保护研究所 | Preparation and regeneration method of biocontrol fungus Acremonium cladosporium protoplast of root-knot nematode |
| CN111705004B (en) * | 2020-07-24 | 2023-04-14 | 天津市农业科学院 | Preparation and regeneration method of biocontrol fungus Acremonium cladosporium protoplast of root-knot nematode |
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