CN101050424A - Method for preparing endophyte of plant - Google Patents
Method for preparing endophyte of plant Download PDFInfo
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- CN101050424A CN101050424A CN 200710065724 CN200710065724A CN101050424A CN 101050424 A CN101050424 A CN 101050424A CN 200710065724 CN200710065724 CN 200710065724 CN 200710065724 A CN200710065724 A CN 200710065724A CN 101050424 A CN101050424 A CN 101050424A
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Abstract
This invention provides a method for preparing endophytes, i.e., enriching endophytic microbes from plant materials. The method comprises: centrifuging plant tissue pulp at a low speed to remove non-pulverized plant cell clusters and part of cell nuclei, further removing impurities such as cell nuclei, organelles and pectin by using haloid salt and surfactant, centrifuging at a high speed, and collecting the precipitate to obtain endophyte samples. It is proven that the enrichment effect of endophytes is obvious as detected by 16S rRNA gene technology of the prokaryotic microbes.
Description
Affiliated field: the present invention relates to a kind of method of microorganism of from vegetable material, giving birth in the enrichment, just relate to a kind of preparation method of endophyte of plant.
Background technology: no matter plant individual is a seed or a towering tree, all is a kind of ecotope of relative complex, and the microorganism that kind is different with abundance perches wherein; For living in plant tissue inside alive plant is not produced the microorganism of direct negative impact, be collectively referred to as endophyte of plant.Endophyte of plant is prevalent in plant materials and of a great variety, be gregarious in some privileged site of plant such as root nodule and the crown-gall nodule except that the small part endophyte is concentrated, most endophyte of plant disperse in plant materials usually and occupy, people to these endophytes perch the position and the kind diversity is known little about it.Moreover, most of environmental microorganism comprises that numerous endophyte of plant is difficult to artificial culture and survives, thereby can not carry out isolation identification and product chemical research by pure culture.In order to use the grand Study on Genome strategy of endophyte to excavate the genetic resources of endophyte of plant and to develop the potential that its compound produces, first step is from the vegetable material enriching plant endophyte and the interference of removing a large amount of plant genetic materials.Relevant report only has the method for enrichment soil microorganisms, its principle is to remove soil impurity by density gradient centrifugation, the inventor once adopted the method for this kind principle but can not effectively be enriched to endophyte from plant tissue, this is because there is the essence difference in the impurity that will remove, and the former is a soil particle and the latter is the plant genetic material.In order to reach from the purpose of vegetable material enrichment endophyte, the present inventor once reported usefulness cellulase and the macerozyme method (Journal of Applied Microbiology 100:830-837) from plant leaf and seed enrichment endophyte in 2006.Follow-up study finds that there is shortcoming in this method, and promptly enzyme action time is longer, may cause enriched sample endophyte abundance deviation because of growing of certain micro-organisms.
Summary of the invention: the objective of the invention is to overcome the limitation of above-mentioned prior art, a kind of new endophyte of plant preparation method is provided.
In order to realize above-mentioned purpose of the present invention, the invention provides following technical scheme:
The preparation method of endophyte of plant, get vegetable material through high-speed tissue mashing machine's homogenate, low-speed centrifugal is got supernatant liquor and is added halogen to final concentration 0.1%～3.0%, is unit with the weight/volume, add tensio-active agent again to final concentration 0.01～2.0%, with the weight/volume is unit, and mixing left standstill 1-2 hour, it is centrifugal to get supernatant liquor, obtains being precipitated as endophyte of plant.
The employed halogen of aforesaid method is preferably used the monovalent cation halogen, or the divalent cation halogen.
The employed tensio-active agent of aforesaid method is preferably with ionogenic surfactant or nonionic surface active agent.
Method of the present invention is based on a certain amount of vegetable material and sterilized water is handled with high-speed tissue mashing machine, homogenate is through the low-speed centrifugal after separating, remove plant origin impurity with certain density halogen and tensio-active agent and comprise nucleus, organoid and colloid etc., get supernatant liquor after the natural subsidence and just can obtain the abundant and very few enriched sample of plant genetic interfering substance of endophyte through the high speed centrifugation collecting precipitation.
Embodiment:
Below in conjunction with the furthermore bright essentiality content of the present invention of embodiments of the invention, but do not limit the present invention with this.
Embodiment 1:
Get trewianudiflora stem skin 40 grams, clean, add 300 ml sterile waters, with the 18000rpm of high-speed tissue mashing machine homogenate with tap water and distilled water; Filter tissue and the fiber of removing not fragmentation with sterile gauze, (200g * 5min) precipitation is removed in separation to filtrate with low-speed centrifugal; Add in the supernatant liquor sodium-chlor to final concentration 1.0% (w/v) and sodium laurylsulfonate to final concentration 0.05% (w/v), put upside down mixing and left standstill 1 hour, have a large amount of flockss and produce this moment, not the disturbance precipitation; The careful new sterilization centrifuge tube of supernatant liquor to of drawing, centrifugal 10 minutes of 5000g, the precipitation that obtains is the endophyte enriched sample.
Embodiment 2:
Get fresh blade 20 grams of Yunnan Caulis Mayteni, clean, add 400 ml sterile waters, with the 15000rpm of high-speed tissue mashing machine homogenate with tap water and distilled water; Filter to remove broken tissue and fiber with sterile gauze, filtrate is with the low-speed centrifugal (precipitation separation of 200g * 5min); Add in the supernatant liquor lithium chloride to final concentration 0.6% (w/v) and Triton X-100 to final concentration 0.15% (w/v), put upside down mixing and left standstill 2 hours, have a large amount of flockss and produce this moment, not the disturbance precipitation; The careful new sterilization centrifuge tube of supernatant liquor to of drawing, centrifugal 10 minutes of 5000g, the precipitation that obtains is the endophyte enriched sample.
Embodiment 3:
Get embodiment 1 or 2 resulting endophyte enriched sample, carry out the detection of endophyte concentration effect.Utilization does not rely on the 16S rRNA gene engineering of cultivation, extracts the DNA of endophyte enriched sample, with the DNA of 2 gram blades or stem skin in contrast, sets up 16S rDNA library; At least 100 clones of picking Pvu II enzyme of carrying out 16S rDNA amplified fragments is cut at random, can not may be derived from prokaryotic micro-organisms by the clone that Pvu II enzyme is cut; This part clone is carried out RFLP somatotype and dna sequencing, infer that according to 16S rDNA sequence this sequence derives from the prokaryotic cell prokaryocyte that plant chloroplast still is an endophyte.Compared with the control, in the enriched sample, the 16S rDNA clone's in prokaryotic micro-organisms source ratio and sequence polymorphism obviously increase (seeing Table 1), illustrate that the inventive method can access rich and varied endophyte of plant, and the plant genetic material disturbs obviously minimizing, thereby has reached from the purpose of vegetable material enrichment endophyte.
The detected result of table 1 endophyte of plant concentration effect of the present invention
| The clone | Blade | The stem skin | ||
| Enrichment | Contrast | Enrichment | Contrast | |
| The clone's number that detects can not be counted the RFLP type by the clone that Pvu II enzyme is cut *Molecular system type procaryotic cell clone ratio | 135 107 20 20pro 79.3 | 110 3 2 2pro 2.7 | 120 97 15 15pro 80.8 | 126 0 0 0 0 |
*Clone's molecular system type is represented the prokaryotic cell prokaryocyte source with pro;
Prokaryotic cell prokaryocyte 16S rDNA clone accounts for total detection clone's percentage ratio.
Claims (3)
1, the preparation method of endophyte of plant, it is characterized in that vegetable material is through high-speed tissue mashing machine's homogenate, low-speed centrifugal is got supernatant liquor and is added halogen to final concentration 0.1%～3.0%, is unit with the weight/volume, add tensio-active agent again to final concentration 0.01～2.0%, with the weight/volume is unit, and mixing left standstill 1-2 hour, it is centrifugal to get supernatant liquor, obtains being precipitated as endophyte of plant.
2, preparation method as claimed in claim 1 is characterized in that halogen preferably uses the monovalent cation halogen, or the divalent cation halogen.
3, preparation method as claimed in claim 1 is characterized in that tensio-active agent is preferably with ionogenic surfactant or nonionic surface active agent.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2007100657244A CN100529051C (en) | 2007-03-19 | 2007-03-19 | Method for preparing endophyte of plant |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2007100657244A CN100529051C (en) | 2007-03-19 | 2007-03-19 | Method for preparing endophyte of plant |
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| Publication Number | Publication Date |
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| CN101050424A true CN101050424A (en) | 2007-10-10 |
| CN100529051C CN100529051C (en) | 2009-08-19 |
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| CNB2007100657244A Expired - Fee Related CN100529051C (en) | 2007-03-19 | 2007-03-19 | Method for preparing endophyte of plant |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101434906B (en) * | 2008-12-19 | 2010-09-15 | 湖南大学 | Separation process for plant endophyte |
| CN101955903A (en) * | 2010-09-28 | 2011-01-26 | 广州市永雄有机肥有限公司 | Bacillus licheniformis bacterial strain and application thereof |
| CN102191190A (en) * | 2010-03-17 | 2011-09-21 | 北京龙裔生物科技有限公司 | Potato with rich SOD complex enzyme and planting method thereof |
| CN103937719A (en) * | 2014-04-22 | 2014-07-23 | 中国科学院新疆生态与地理研究所 | Method for separating endophytic bacteria of euhalophyte |
| CN112795513A (en) * | 2021-02-03 | 2021-05-14 | 南京翠京元生物科技有限公司 | Enrichment and separation method for endophyte |
| CN113913411A (en) * | 2021-12-01 | 2022-01-11 | 北京林业大学 | Composition for extracting fungal protoplast from plant and application thereof |
-
2007
- 2007-03-19 CN CNB2007100657244A patent/CN100529051C/en not_active Expired - Fee Related
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101434906B (en) * | 2008-12-19 | 2010-09-15 | 湖南大学 | Separation process for plant endophyte |
| CN102191190A (en) * | 2010-03-17 | 2011-09-21 | 北京龙裔生物科技有限公司 | Potato with rich SOD complex enzyme and planting method thereof |
| CN101955903A (en) * | 2010-09-28 | 2011-01-26 | 广州市永雄有机肥有限公司 | Bacillus licheniformis bacterial strain and application thereof |
| CN101955903B (en) * | 2010-09-28 | 2012-06-27 | 广州市永雄有机肥有限公司 | Bacillus licheniformis bacterial strain and application thereof |
| CN103937719A (en) * | 2014-04-22 | 2014-07-23 | 中国科学院新疆生态与地理研究所 | Method for separating endophytic bacteria of euhalophyte |
| CN103937719B (en) * | 2014-04-22 | 2016-01-27 | 中国科学院新疆生态与地理研究所 | A kind of method being separated euhalophyte endogenetic bacteria |
| CN112795513A (en) * | 2021-02-03 | 2021-05-14 | 南京翠京元生物科技有限公司 | Enrichment and separation method for endophyte |
| CN113913411A (en) * | 2021-12-01 | 2022-01-11 | 北京林业大学 | Composition for extracting fungal protoplast from plant and application thereof |
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| Publication number | Publication date |
|---|---|
| CN100529051C (en) | 2009-08-19 |
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Granted publication date: 20090819 Termination date: 20130319 |