CN104450671A - Breeding method of fast-growing type Frankia bacterial strain - Google Patents
Breeding method of fast-growing type Frankia bacterial strain Download PDFInfo
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- CN104450671A CN104450671A CN201410680717.5A CN201410680717A CN104450671A CN 104450671 A CN104450671 A CN 104450671A CN 201410680717 A CN201410680717 A CN 201410680717A CN 104450671 A CN104450671 A CN 104450671A
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Abstract
The invention discloses a breeding method of a fast-growing type Frankia bacterial strain. The breeding method comprises the following steps: sterilizing Hippophae nodules, then putting in a mutagenic culture medium prepared in advance, and then homogenizing to prepare bacterial suspension liquid, wherein the contents of phosphorus, boron, magnesium and hydrolysis casein in the mutagenic culture medium are higher; adding a mutagenic agent to the bacterial suspension liquid prepared in the first step for treatment for 50 min, then putting under a 20W ultraviolet lamp for irradiation for 300 s; carrying out fermental cultivation on the bacterial suspension liquid through mutagenic treatment in the second step by utilizing 96-well plates; carrying out high-throughput screening by utilizing a rapid bioassay method, and then obtaining a mutant strain through three-wheel secondary screening, wherein the breeding speed of the mutant strain is 4-12 times higher than that of an original strain, and the mutant strain is a fast growing strain. According to the breeding method disclosed by the invention, rapid-growing type Frankia bacteria are obtained based on the principle of induced mutation, the method is simple and easy to operate, slow-growing type Frankia bacteria can be induced into fast-growing bacteria, and the blank that the rapid-growing type Frankia bacteria are difficult to obtain in the prior art is filled.
Description
Technical field
The present invention relates to a kind of method for strain breeding thereof, be specifically related to a kind of selection of fast-growing type Frankia Salmonella bacterial strain.
Background technology
Molecular nitrogen (N
2) in air stratosphere, content accounts for 78%, but plant can not utilize, and needs them to be converted into NH
4 +or NO
3 -rear plant just can absorb.By molecular nitrogen (N on the earth
2) be converted into NH
4 +biology only have 3 class floras in microorganism: azotobacter, combination azotobacter and symbiotic nitrogen-fixing bacteria, they all belong to prokaryotic organism; Azotobacter and combination azotobacter nitrogen-fixing efficiency very low, symbiotic nitrogen-fixing bacteria comprises again legume nodule bacterium and non-leguminous plant actinomycetes Frankia Salmonella.
The nitrogen-fixing efficiency of Frankia bacterium is the highest, accounts for more than 80% of symbiotic nitrogen fixation total amount, such as: be 85-300kgN with alder symbiotic nodulation and nitrogen fixation amount
2/ hm
2/ y, red branch alder is 140-209, and autumn alder is 40-157, and Horsetail Beefwood is 229, and sea-buckthorn is 47-179, and Coriaria sinica is 150, and the amount of nitrogen fixation of root nodule bacteria only has 13.3kgN
2/ hm
2/ y, the former nitrogen fixing capacity higher than the latter 3-10 doubly more than.So research Forlan kirschner symbiotic nitrogen-fixing bacteria, develops novel microorganism fertilizer and has more huge ecology, environment benefits and economic gains.
But in the last hundred years, Frankia bacterium artificial culture does not succeed.Until American scientist Callaham first time in 1978 obtains Frankia bacterium from wooden root nodule fainted by perfume (or spice), and after artificial culture success, the formal research and development to Frankia bacterium open road.
But over nearly more than 30 years, countless scientist uses multiple substratum, cultural method, just find after separation and Culture, Frankia bacterium is a class " slowly raw type " flora.Artificial plate culture medium is cultivated and within about 40 days, the small bacterium colony of white could be seen with magnifying glass, several grams of bacterium powder for research be expected, very time-consuming, bothersome, effort, difficulty are very large.Therefore, scientific circles still rest on ecology, classification, system, physiology, biochemistry, heredity, plasmid, primed probe, molecular hybridization scheduling theory aspect to the research of Frankia Salmonella both at home and abroad at present, and applied research almost still belongs to blank out, let alone batch fermentation is applied to and produces.
Therefore, need a kind of method now, the bacterium that " will give birth to type slowly ", mutagenesis is domesticated for " soon raw bacterium ".
Summary of the invention
For solving the problems of the technologies described above, the object of the present invention is to provide and a kind ofly raw type Frankia Salmonella mutagenesis can tame into the selection of fast raw bacterium slowly.
For achieving the above object, technical scheme of the present invention is as follows:
The selection of fast-growing type Frankia Salmonella bacterial strain, comprises the following steps:
The first step, preparation bacterium suspension: first by old for sea-buckthorn root nodule sterilization, and then be transferred in the Induced medium of preparation in advance, then bacterium suspension is made in homogenate, wherein, the phosphorus of described Induced medium, boron, magnesium and hydrolysis network protein content are higher;
Second step, mutagenic treatment: add mutagenic compound process 50min in the bacterium suspension prepared in a first step, and then irradiate 300s under proceeding to 20W ultraviolet lamp;
3rd step, fermentation culture: the bacterium suspension through mutagenic treatment in described second step is utilized 96 orifice plate fermentation culture;
4th step, screening: utilize quick bio assay method to carry out high flux screening, then take turns multiple sieve by 3, obtains mutant strain, faster than the reproduction speed of starting strain 4 ~ 12 times of described mutant strain, and described mutant strain is fast raw bacterium.
Preferably, also comprise and shaking table cultivation screening is carried out to described fast raw bacterium, the described fast raw bacterium obtained is inoculated in screening culture medium, be placed on the shaking table of rotating speed 140 ~ 200rpm/min, culture temperature is 24 ~ 30 DEG C, cultivate 10 days, described fast raw bacteria strain breeding coefficient turn improves 1 ~ 1.5 times, and dry mycelium yield is 2.54%.
Preferably, described screening and culturing based component comprises following several: potassium primary phosphate, potassium hydrogen phosphate, magnesium sulfate, calcium chloride, propionic salt, hydrolysis network albumen, potato saccharified liquid, liquid microelement, VITAMIN, Fe-Na-EDTA salt, purified water, regulates described screening culture medium pH to be 7.2.
Preferably, described Induced medium composition comprises following several: potassium primary phosphate, potassium hydrogen phosphate, magnesium sulfate, calcium chloride, borate, hydrolysis network albumen, sucrose, liquid microelement, VITAMIN, Fe-Na-EDTA salt, purified water, regulates described screening culture medium pH to be 7.2.
Preferably, in the first step by old for sea-buckthorn root nodule 0.1%HgCl
2vacuum-infiltration is sterilized.
Preferably, the mutagenic compound described in second step are the mutagenic compound containing 2.5mg/mL nitrosoguanidine.
Preferably, when using ultra violet lamp in second step, ultraviolet lamp is 25cm from the distance of bacterium suspension.
The invention has the beneficial effects as follows:
One, selection disclosed by the invention, it is the principle acquisition fast-growing type Frankia Salmonella based on Mutation induction, the method is simple to operation, can tame into fast raw bacterium, having filled up in prior art the blank being difficult to obtain fast-growing type Frankia Salmonella by giving birth to slowly the mutagenesis of type Frankia Salmonella.
Two, the inventive method, adopt 2.5mg/mL nitrosoguanidine to be mutagenic compound, mutation efficiency is high, and coordinates culture medium culturing, and the pick-up rate of fast-growing type Frankia Salmonella is higher.
Three, selection disclosed by the invention, can obtain fast-growing type Frankia Salmonella efficiently, for exploitation has established good basis about Frankia Salmonella novel microorganism fertilizer, has good ecological, environmental protective and economic benefit.
Accompanying drawing explanation
In order to be illustrated more clearly in the technical scheme in embodiment of the present invention technology, be briefly described to the accompanying drawing used required in the description of embodiment technology below, apparently, accompanying drawing in the following describes is only some embodiments of the present invention, for those of ordinary skill in the art, under the prerequisite not paying creative work, other accompanying drawing can also be obtained according to these accompanying drawings.
Fig. 1 is the MFz8 that the present invention cultivates 20h
#(changing former numbering into existing numbering, so that consistent with culture presevation numbering) bacterium bacterium colony.
Fig. 2 is MFz8 of the present invention
#the mycelium of bacterium.
Fig. 3 is MFz8 of the present invention
#mycelia and vesicle.
Fig. 4 is MFz8 of the present invention
#mycelia and spore.
Wherein, mycelia is the body that grows, and vesicle is fixed nitrogen structure, and containing nitrogenase, spore is propagulum.
Embodiment
Below in conjunction with the accompanying drawing in the embodiment of the present invention, be clearly and completely described the technical scheme in the embodiment of the present invention, obviously, described embodiment is only the present invention's part embodiment, instead of whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art, not making the every other embodiment obtained under creative work prerequisite, belong to the scope of protection of the invention.
Embodiment 1
First prepare Induced medium according to the formula of Induced medium, Induced medium composition comprises following several: potassium primary phosphate, potassium hydrogen phosphate, magnesium sulfate, calcium chloride, borate, hydrolysis network albumen, sucrose, liquid microelement, VITAMIN, Fe-Na-EDTA salt, purified water, regulates described screening culture medium pH to be 7.2.
Formula according to screening culture medium prepares screening culture medium, screening and culturing based component comprises following several: potassium primary phosphate, potassium hydrogen phosphate, magnesium sulfate, calcium chloride, propionic salt, hydrolysis network albumen, potato saccharified liquid, liquid microelement, VITAMIN, Fe-Na-EDTA salt, purified water, regulates described screening culture medium pH to be 7.2.
Then comprise following two according to mutagenesis cultivation and shaking table cultivation screening to walk greatly, tool method, comprises the following steps:
The first step, preparation bacterium suspension: first by old for sea-buckthorn root nodule 0.1%HgCl
2vacuum-infiltration is sterilized, and then is transferred in the Induced medium of above-mentioned preparation, and then bacterium suspension is made in homogenate.
Second step, mutagenic treatment: add the mutagenic compound containing 2.5mg/mL nitrosoguanidine in the bacterium suspension prepared in a first step, process 50min, and then irradiate 300s under proceeding to 20W ultraviolet lamp, ultraviolet lamp is 25cm from the distance of bacterium suspension.
3rd step, fermentation culture: the bacterium suspension through mutagenic treatment in described second step is utilized 96 orifice plate fermentation culture.
4th step, screening: utilize quick bio assay method to carry out high flux screening, then take turns multiple sieve by 3, obtains mutant strain, faster than the reproduction speed of starting strain 4 ~ 12 times of described mutant strain, and described mutant strain is fast raw bacterium.
5th step, shaking table is carried out to described fast raw bacterium and cultivates screening, the described fast raw bacterium obtained is inoculated in screening culture medium, be placed on the shaking table of rotating speed 140 ~ 200rpm/min, culture temperature is 24 ~ 30 DEG C, cultivate 10 days, described fast raw bacteria strain breeding coefficient turn improves 1.2 times, and dry mycelium yield is 2.54%.
In the present embodiment, take label as the bacterial strain of MFz8 be example, MFz8 bacterial strain breeding coefficient improves nearly 12 times (as Fig. 1,2).
Embodiment 2
In embodiment 2, after obtaining fast raw bacterium, shaking table is carried out to fast raw bacterium and cultivates screening, the described fast raw bacterium obtained is inoculated in screening culture medium, be placed on the shaking table of rotating speed 160 ~ 180rpm/min, culture temperature is 26 ~ 28 DEG C, cultivates 10 days, described fast raw bacteria strain breeding coefficient turn improves 1.5 times, and dry mycelium yield is 2.96%.
Its cultivation results as shown in Figures 3 and 4.
Consistent all with embodiment 1 of other step in embodiment 2 and mutagenesis culture condition.
Visible embodiment 2, when other condition is constant, changes culture temperature and shaking speed, also affects to some extent the fast raw bacterium cultivation results obtained.
Telling somebody what one's real intentions are in book, mentioning by selecting different substratum finally to determine best, if the formula enumerating several substratum in experimentation can be asked
Illustrate: at Frankia, raw bacterium mutagenesis is in fast raw bacterium mutagenesis cultivating process slowly, and we explored and minimumly used 10 kinds of substratum nearly.These the two kinds of Induced mediums more than reported, screening culture medium are two kinds of substratum that best last success ratio is the highest.
" China Committee for Culture Collection of Microorganisms's common micro-organisms " center " preservation; depositary institution address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City; preservation date: on October 15th, 2014; deposit number: 9769, Classification And Nomenclature: Frankia brunchorst that this biomaterial is delivered to.
To the above-mentioned explanation of the disclosed embodiments, professional and technical personnel in the field are realized or uses the present invention.To be apparent for those skilled in the art to the multiple amendment of these embodiments, General Principle as defined herein can without departing from the spirit or scope of the present invention, realize in other embodiments.Therefore, the present invention can not be restricted to these embodiments shown in this article, but will meet the widest scope consistent with principle disclosed herein and features of novelty.
Claims (7)
1. the selection of fast-growing type Frankia Salmonella bacterial strain, is characterized in that, comprise the following steps:
The first step, preparation bacterium suspension: first by old for sea-buckthorn root nodule sterilization, and then be transferred in the Induced medium of preparation in advance, then bacterium suspension is made in homogenate, wherein, the phosphorus of described Induced medium, boron, magnesium and hydrolysis network protein content are higher;
Second step, mutagenic treatment: add mutagenic compound process 50min in the bacterium suspension prepared in a first step, and then irradiate 300s under proceeding to 20W ultraviolet lamp;
3rd step, fermentation culture: the bacterium suspension through mutagenic treatment in described second step is utilized 96 orifice plate fermentation culture;
4th step, screening: utilize quick bio assay method to carry out high flux screening, then take turns multiple sieve by 3, obtains mutant strain, faster than the reproduction speed of starting strain 4 ~ 12 times of described mutant strain, and described mutant strain is fast raw bacterium.
2. the selection of fast-growing type Frankia Salmonella bacterial strain according to claim 1, it is characterized in that, also comprise and shaking table cultivation screening is carried out to described fast raw bacterium, the described fast raw bacterium obtained is inoculated in screening culture medium, be placed on the shaking table of rotating speed 140 ~ 200rpm/min, culture temperature is 24 ~ 30 DEG C, cultivates 10 days, described fast raw bacteria strain breeding coefficient turn improves 1 ~ 1.5 times, and dry mycelium yield is 2.54%.
3. the selection of fast-growing type Frankia Salmonella bacterial strain according to claim 2, it is characterized in that, described screening and culturing based component comprises following several: potassium primary phosphate 1g/L, dipotassium hydrogen phosphate 1g/L, magnesium sulfate 1g/L, calcium chloride 0.025g/L, Sodium Propionate 0.02g/L, hydrolysis network albumen 4g/L, potato saccharified liquid 6g/L, liquid microelement 3ml, VITAMIN 2ml, Fe-Na-EDTA salt 2ml, purified water, regulate described screening culture medium pH to be 7.2.
4. the selection of fast-growing type Frankia Salmonella bacterial strain according to claim 1, it is characterized in that, described Induced medium composition comprises following several: sucrose 5g, potassium primary phosphate 1.5g/L, dipotassium hydrogen phosphate 1.5g/L, magnesium sulfate 1g/L, calcium chloride 0.05g/L, Sodium Propionate 0.02g/L, hydrolysis network albumen 4g/L, potato saccharified liquid 6g/L, liquid microelement 3ml, VITAMIN 2ml, Fe-Na-EDTA salt 2ml, purified water, regulate described screening culture medium pH to be 7.2.
5. the selection of fast-growing type Frankia Salmonella bacterial strain according to claim 1, is characterized in that, by old for sea-buckthorn root nodule 0.1%HgCl in the first step
2vacuum-infiltration is sterilized.
6. the selection of fast-growing type Frankia Salmonella bacterial strain according to claim 1, it is characterized in that, the mutagenic compound described in second step are the mutagenic compound containing 2.5mg/mL nitrosoguanidine.
7. the selection of fast-growing type Frankia Salmonella bacterial strain according to claim 6, is characterized in that, when using ultra violet lamp in second step, ultraviolet lamp is 25cm from the distance of bacterium suspension.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104446694A (en) * | 2014-11-25 | 2015-03-25 | 张吉科 | Frankia super bacterial manure fermenting technology |
| CN108531428A (en) * | 2018-04-16 | 2018-09-14 | 中科国思生物科技研究（广州）有限公司 | A kind of preparation method of fast-growing frankia bacterium powder |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN86107988A (en) * | 1985-12-30 | 1987-12-02 | 斯文-埃里克·尼尔森 | Biological nitrogen fixation in the non-leguminous plant |
| CN101899430A (en) * | 2010-07-12 | 2010-12-01 | 甘肃农业大学 | High-efficiency nitrogen-fixing microorganism mutation breeding method |
-
2014
- 2014-11-25 CN CN201410680717.5A patent/CN104450671A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN86107988A (en) * | 1985-12-30 | 1987-12-02 | 斯文-埃里克·尼尔森 | Biological nitrogen fixation in the non-leguminous plant |
| CN101899430A (en) * | 2010-07-12 | 2010-12-01 | 甘肃农业大学 | High-efficiency nitrogen-fixing microorganism mutation breeding method |
Non-Patent Citations (2)
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| 张吉科 林纬: "沙棘根瘤的形成与固氮能力", 《沙棘》 * |
| 赵沫 等: "高产花生四烯酸菌的原生质体诱变育种研究", 《食品科学》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104446694A (en) * | 2014-11-25 | 2015-03-25 | 张吉科 | Frankia super bacterial manure fermenting technology |
| CN108531428A (en) * | 2018-04-16 | 2018-09-14 | 中科国思生物科技研究（广州）有限公司 | A kind of preparation method of fast-growing frankia bacterium powder |
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