CN102965296A - Nitrogen-fixing Klebsiella sp. with high anti-glyphosate activity and its application - Google Patents
Nitrogen-fixing Klebsiella sp. with high anti-glyphosate activity and its application Download PDFInfo
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- CN102965296A CN102965296A CN2012100298276A CN201210029827A CN102965296A CN 102965296 A CN102965296 A CN 102965296A CN 2012100298276 A CN2012100298276 A CN 2012100298276A CN 201210029827 A CN201210029827 A CN 201210029827A CN 102965296 A CN102965296 A CN 102965296A
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Abstract
The invention relates to a Klebsiella sp. strain with a nitrogen-fixing effect and high anti-glyphosate capability and its application. The strain is obtained from sugarcane rhizosphere separation. After nitrogen-free culture medium screening, nitrogenase activity determination, nitrogenase gene nif H amplification and sequence analysis, the strain is determined as an associative nitrogen-fixing bacterium. And by strain morphology and of 16S rDNA sequence analysis, the strain is identified as a Klebsiella sp. bacterium. Named as GG08160, the strain provided in the invention has an associative nitrogen-fixing capability and an inorganic phosphorus degradation function, can significantly promote sugarcane seedling growth, and can still grow normally in a culture medium with a glyphosate content up to 250mM. With high anti-glyphosate activity and good application prospects, the strain can serve as a plant growth-promoting bacterium, and also provides a new microorganism resource or gene resource for study of glyphosate biodegradation, as well as provides resources and support for promoting agricultural sustainable development.
Description
Technical field
The present invention relates to biological technical field, relate to particularly Cray Bai Shi bacterium and application thereof that a strain has nitrogen fixation and high-resistance glyphosate ability.
Background technology
Give full play to the effect of agriculture Microbial resources treasure-house, actively excavating new Biocontrol microorganism resource is the important content of agriculture Microbial resources development research.In recent years, use in a large number the wasting of resources that chemical fertilizer, agricultural chemicals cause and soil, body of groundwater pollutes and has caused the extensive attention of countries in the world.Utilizing biological nitrogen fixation is to reduce the most effectively approach of amount of application of nitrogen fertilizer, and the research of biological nitrogen fixation resource, development and utilization occupy critical role in China's agricultural sustainable development.Though association nitrogen fixation efficient is high not as good as symbiotic nitrogen fixation, it distributes wide, and the crop that is benefited is many, thereby is the very important fixed nitrogen system of a class.Found that at present great majority have the grasses such as the crop of important value such as sugarcane, paddy rice, wheat, corn, herbage and all can carry out associative nitrogen fixation with vinelandii.Combination azotobacter is except also having simultaneously secretion growth hormone, molten phosphorus for the host provides the nitrogen, strengthening the effect of many-sided Promoting plant growths such as plant disease resistance, degeneration-resistant border.The soil fertility of bringing for long-term single administration chemical fertilizer descends, quality of agricultural product descends and a series of negative impacts to agricultural sustainable development such as agricultural environment pollution aggravation, actively excavates and utilizes the potentiality of associative nitrogen fixation that agricultural sustainable development is had more special meaning.
Glyphosate is a kind of inner sucting conduction type, and the wide spectrum steriland herbicide is annual to kind more than 40, perennial, unifacial leaf and dicotyledonous woody, herbaceous plant has lethal effect, and is especially strong to the underground organization destructive force of perennial grass, is widely used in the rubber plantation, fruit tree, tea place, mulberry field, sugarcane field and forestry weeding.The glyphosate physico-chemical property is stable, have efficient, wide spectrum, low toxicity, low residue, not the spoiled soil environment, most plants had the incomparable advantage of other weedicide such as the natural disposition of going out, be one of kind of whole world weedicide usage quantity maximum.But glyphosate has killing effect equally to farm crop when killing weeds, thereby has limited its use range and duration of service.Glyphosate enters in the plant materials, suppresses 5 one enol acetonyl shikimic acids, one 3 one phosphate synthases (EPSP synthase), makes plant can not synthesize necessary some aromatic amino acid of its existence, causes plant dead.Do not need synthetic those aromatic amino acids and people and animal are different from the metabolism of plant, so glyphosate is to people and animal safety.
Biological degradation is the main approach of glyphosate degraded.Many microorganisms have Degradation to glyphosate, and the main intermediate product of glyphosate microbiological deterioration is the extremely low aminomethyl phosphonic acid of toxicity (AMPA), finally is produced as water, carbonic acid gas and phosphoric acid.In physical environment particularly in the soil in the weedicide areas such as life-time service glyphosate, exist miscellaneous can tolerance or the bacterium of degradation of glyphosate.The research of individual plant degradation by bacteria glyphosate all is separated to the glyphosate degradation bacteria at Rhizobiaceae, Arthrobacter, klebsiella pneumoniae etc. from early eighties.In soil, rubbish and water body example, also can be separated to the bacterial isolates of high resistance or degradation of glyphosate.If change farm crop over to after will deriving from the glyphosate resistance gene clone of microorganism, can strengthen plant to the resistance of glyphosate, or increase the ability of the sweet phosphine of plant degradation, thereby can remove glyphosate to the harm of farm crop.If can there be the farm crop of high resistance or degradation of glyphosate to occur, then can save widely farm production cost, can greatly promote the glyphosate industrial expansion again.
Summary of the invention
The object of the invention is for practical problems and the needs of agricultural production practice, filter out sugarcane production is had remarkable promoter action, the combination azotobacter that simultaneously glyphosate is had highly-resistant activity is for research, the application of developing short sugarcane production microbiobacterial agent and resistance glyphosate genetically modified crops provides new Microbial resources.
The technical scheme that the present invention takes
Azotobacter belongs to klebsiella (Klebsiella sp.) in the sugarcane of the present invention, this bacterial strain has been stored in (address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), China Committee for Culture Collection of Microorganisms common micro-organisms center on November 3rd, 2011, be called for short CGMCC, deposit number is CGMCC No.5439.
This bacterial strain separates acquisition by Among the Sugarcane root, by nitrogen-free agar screening, nitrogenase activity determination, nifHDK nifH amplification and sequential analysis, determines that it is combination azotobacter; Through strain morphology, 16S rDNA sequential analysis, be this identification of strains Klebsiella (Klebsiella sp.) bacterium.Bacterial strain called after GG08160, its optimum growth temperature is 30 ℃, the most suitable growth pH is 7.0～7.2.
Bacterial strain GG08160 of the present invention has the function of associative nitrogen fixation ability and degraded inorganic phosphorus, growth has remarkable promoter action to Sugarcane Seedlings, reaching in the culture medium of 250mM still at glyphosate content can normal growth, activity with high-resistance glyphosate, have a good application prospect, can be used as the short living bacterium of plant-growth, the biological degradation research that can be simultaneously glyphosate provides new Microbial resources or genetic resources, for promoting that agricultural sustainable development provides resource and guarantee.
Description of drawings
The growing state of Fig. 1 bacterial strain GG08160 in different concns glyphosate liquid nutrient medium;
The growing state of Fig. 2 bacterial strain GG08160 on Pikovaskaia ' s culture medium flat plate;
Fig. 3 bacterial strain GG08160 is to the promoter action (increasing the dry weight of plant) of sugar-cane tissue culture seedlings growth;
Fig. 4 bacterial strain GG08160 is to the promoter action (increase plant nitrogen content) of sugar-cane tissue culture seedlings growth;
Fig. 5 bacterial strain GG08160 is to the promoter action (plant growing way and association nitrogen fixation efficient) of sugar-cane tissue culture seedlings growth.
Embodiment
Following examples are used for explanation the present invention.
The separation of embodiment 1 bacterial strain of the present invention
Take by weighing and be attached to sugarcane root table soil 5g and put into the 45mL aqua sterilisa, 180r/min on the shaking table, 28 ℃ of constant temperature oscillation 30min disperse microorganism cellss, leave standstill 10min, namely get 10
-1Diluent with aqua sterilisa 10 times of serial dilutions, is respectively got 100 μ L and is coated in the Ashby substratum (composition is sucrose 10g, NaCl 0.2g, KH
2PO
40.2g, MgSO
47H
2O 0.2g, CaSO
42H
2O 0.1g, CaCO
35g, distilled water 1000mL, pH 7.0) on the flat board, cultivate 2-3d to growing single bacterium colony at 28 ℃.With the different single bacterium colonies of inoculating needle picking, obtain single bacterium colony 3 times at Ashby flat board line purifying.Single colony inoculation behind the purifying on LB substratum (composition is Tryptones 10g, yeast extract 5g, sodium-chlor 10g, distilled water 1000mL, pH 7.0) flat board, is deposited for 4 ℃, in the recent period; Single colony inoculation of purifying is that 15% glycerine mixes in the LB liquid culture to logarithmic phase with bacterium liquid and final concentration ,-80 ℃ of refrigerator prolonged preservation.
The nitrogenase activity of embodiment 2 bacterial strains of the present invention
Adopt Acetylene Reduction ethylene process (acetylene reduction assay, ARA) to measure nitrogenase activity.With the 48h that in the Ashby liquid nutrient medium, grows after the GG08160 activation, get in the 60mL ground narrow-mouth bottle that contains 10mL Ashby liquid nutrient medium that 1mL bacterium liquid is inoculated in sterilization, in bottle, extract 5mL gas out after cultivating 24h, and then injection 5mL acetylene gas, continue to cultivate 24h, detect the ethene growing amount with gas chromatograph, with nmolC
2H
4/ h.mL represents nitrogenase activity.With the positive contrast of azotobacter G.diazotrophicus PAL5 in the Brazilian sugarcane, with the processing of inoculation 1mL inactivated bacterial liquid as blank.
Experimental result shows that the Acetylene Reduction ethene activity of the nitrogenase of bacterial strain of the present invention is 1632nmol C
2H
4/ h is with the nitrogenase activity 1675nmol C of azotobacter type strain G.diazotrophicus PAL5 in the Brazilian sugarcane of positive control
2H
4/ h is suitable.
The nifH gene of embodiment 3 bacterial strains of the present invention and the clonal analysis of 16S rDNA
The preparation of pcr amplification template: will be inoculated into LB liquid nutrient medium (composition is Tryptones 10g, yeast extract 5g, sodium-chlor 10g, distilled water 1000mL, pH 7.0) after the bacterial strain GG08160 activation, cultivate 20h after centrifugal collection thalline for subsequent use.With bacterial genomes DNA extraction test kit, press product description extracts bacterial strain of the present invention from fresh bacterium LB culture genomic dna, be stored in-20 ℃, dna content is about 50-100ng/ μ L.
The nifH genetic analysis: amplimer is PolyF (TGCGAYCCSAARGCBGACTC) and PolyR (ATSGCCATCATYTCRCCGGA), the PCR response procedures is: 94 ℃ of 3min denaturations, then carry out 94 ℃ of 1min, 55 ℃ of 1min, 72 ℃ of 1min, 30 circulations, last 72 ℃ of 10min.Pcr amplification product carries out 1.5% agarose gel electrophoresis and detects.With the positive contrast of fixed nitrogen glucose vinegar acidfast bacilli G.diazotrophicus PAL5 bacterial strain (Gillis et al., 1989), with the negative contrast of intestinal bacteria E.coli DH5 α bacterial strain.Be cloned on the T carrier after the nifH amplified production reclaimed, and clone test kit by the pMDTM18-T Vector of precious biotechnology (Dalian) company limited (being called for short TaKaRa) and illustrate to operate.Serve the sea through the positive colony of PCR checking and give birth to the order-checking of worker bio-engineering corporation, sequencing result is at GenBank login and compare of analysis.
The 16S rDNA sequential analysis of bacterial strain: the 16S rRNA gene that utilizes bacterial 16 S universal primer 27F (AGAGTTTGATCCTGGCTCAG) and 1492R (GGTTACCTTGTTACGACTT) amplification bacterium.The PCR response procedures is: 94 ℃ of 3min denaturations, then carry out 94 ℃ of 55s, 50 ℃ of 50s, 72 ℃ of 1min, 35 circulations, last 72 ℃ of 10min.Pcr amplification product carries out 1.2% agarose gel electrophoresis and detects.The PCR product is cloned on the T carrier after reclaiming, and selects positive colony to serve the sea and gives birth to the order-checking of worker bio-engineering corporation, and sequencing result is at GenBank login and compare of analysis.
The sequencing fragment analytical results that the nifH pcr amplification obtains shows that the GG08160 bacterial strain contains molybdenum-iron Fe protein of nitrogenase gene nifH sequence (accession number FJ823000); 16S rDNA the sequencing results (accession number FJ823051) shows that the homology of bacterial strain of the present invention and Klebsiella (Klebsiella sp.) bacterium reaches 98%.The sequencing results of colonial morphology, nifH gene and the 16S rDNA of comprehensive bacterial strain is Klebsiella (Klebsiella sp.) bacterium with identification of strains of the present invention.
Embodiment 4 bacterial strains of the present invention are measured the tolerance of glyphosate
Take LB as basic medium, in the LB liquid nutrient medium, add ammonium glyphosate (available from the autonomous chemical industry in Guangxi company limited), preparation contains the serial culture base that the glyphosate final concentration is respectively 0,50,100,150,200,250,300mM.
Bacterial strain GG08160 is inoculated into the LB liquid culture based on 30 ℃, and 180rpm cultivates 24h as mother liquor, get 20 μ L mother liquors and add in the above-mentioned substratum that contains the different concns glyphosate of 50mL, 30 ℃, cultivate 48h under the 180rpm condition after, measure the OD of nutrient solution
600
Measurement result is seen Fig. 1, and GG08160 has stronger tolerance to glyphosate, reaches in glyphosate concentration on the substratum of 250mM to keep good growth, cultivates at the 300mM glyphosate and still has a small amount of growth.
Embodiment 5 bacterial strains of the present invention are to the degradation characteristic of inorganic phosphorus
Get 1ml bacterium liquid after the bacterial strain activation culture to be measured, the centrifugal 5min of 10000rpm removes supernatant, then washes bacterium 2 times with the centrifugal 5min of aqua sterilisa 10000rpm, obtains bacteria suspension with 10 times of aqua sterilisa dilutions.Getting strains tested bacteria suspension 10 μ l points is inoculated in and fills Pikovaskaia ' s substratum (composition: glucose 10g, (NH
4)
2SO
40.5g, NaCl 0.3g, KCl 0.3g, MgSO
47H
2O 0.3g, MnSO
44H
2O 0.03g, FeSO
47H
2O 0.03g, Ca
3(PO
4)
25g, agar powder 15g, distilled water 1000mL, pH 7.0) culture dish in, repeat 4 times, place 28 ℃ of lower 3d of cultivation.Have or not transparent circle to form around observing inoculating strain, measure the size of transparent circle diameter (D) and colony diameter (d), judge that according to the ratio size of the transparency D/d of molten phosphorus circle bacterium phosphorus decomposing ability is strong and weak.
Experimental result is seen Fig. 2, and the GG08160 bacterial strain can form transparent circle at Pikovaskaia ' s plate culture medium, shows that this bacterial strain can secrete some acidic substance, and the diffusion of peripherad substratum, makes the phosphoric acid salt [Ca of periphery of bacterial colonies
3(PO
4)
2] dissolving, namely this bacterial strain has degraded inorganic phosphorus characteristic, and the molten phosphorus loop diameter D (bacterium colony+transparent circle) of bacterial strain is 1.32 with the ratio (D/d) of colony diameter (d).
Embodiment 6 bacterial strains of the present invention are to the promoter action of sugar-cane tissue culture seedlings growth
Strains tested is respectively azotobacter G.diazotrophicus PAL5 bacterial strain in GG08160 and the Brazilian sugarcane.With the cultivation matrix after the transplantation of seedlings of vermiculite sugarcane tissue-culture, in proportion every kg matrix add 10mg (
15NH
4)
2SO
4(
15N atom percentage surpasses 10.11), divide to install in the plastic tub.
After strains tested activated respectively on the LB liquid nutrient medium overnight incubation, centrifugal collection thalline is diluted to every milliliter of 1-2 * 10 with after the aqua sterilisa suspended centrifugal cleaning 2 times
8Individual cell.The consistent sugar-cane tissue culture seedlings of growth after the hardening is transplanted in the plastic tub that cultivation matrix is housed, and every basin pours into the bacteria suspension 100mL of above-mentioned strains tested GG08160, places 28 ℃, photoperiod 14h light-10h half-light photograph incubator cultivation.Fill with the root inoculation with the bacteria suspension of the same concentration of 100mL again behind the 10d, continue under limit bacterium condition, to cultivate.With the positive contrast of bacteria suspension of the PAL5 bacterial strain of inoculation equivalent same concentration, blank inoculation equivalent aqua sterilisa.Growing state to sugar-cane tissue culture seedlings behind the 50d carries out record, and the results plant is also cleaned cultivation matrix, and mensuration plant dry weight, nitrogen content reach after the lyophilize
15N content calculates nitrogen-fixing efficiency.Nitrogen-fixing efficiency is according to formula Ndfa=100 * [1-(atom%
15N inoculates plant/atom%
15The N adjoining tree)] calculate.
Test determination is the result show: inoculation GG08160 can promote significantly that (Fig. 3-Fig. 5), the dry weight of sugar-cane tissue culture seedlings and total nitrogen content have increased by 42.56% and 38.64% (Fig. 3, Fig. 4) than blank respectively for the growth of sugar-cane tissue culture seedlings; Plant obtains percentage (nitrogen-fixing efficiency) average out to 9.75% (Fig. 5) of nitrogen from air; And dry weight and the total nitrogen content rate of increase of the sugar-cane tissue culture seedlings of inoculation PAL5 bacterial strain are respectively 29.35% and 35.25%, and nitrogen-fixing efficiency is 10.09%.
Claims (1)
1. a strain has the fixed nitrogen klebsiella of highly-resistant activity to glyphosate, it is characterized in that: bacterial strain called after GG08160, have the activity of high-resistance glyphosate, and can and significantly promote the growth of Sugarcane Seedlings with the sugarcane association nitrogen fixation.
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| CN103911317A (en) * | 2014-03-07 | 2014-07-09 | 中国农业科学院生物技术研究所 | Bacteria capable of resisting glyphosate, and applications thereof |
| CN104351255A (en) * | 2014-11-10 | 2015-02-18 | 云南农业大学 | Application of Klebsiella variicola SH-1 strain |
| CN105063166A (en) * | 2015-09-16 | 2015-11-18 | 东莞市保得生物工程有限公司 | Nitrogen-free solid medium and method for judging ammonia secretion of free living nitrogen fixing bacteria by utilizing solid medium |
| CN105754901A (en) * | 2016-04-13 | 2016-07-13 | 西北农林科技大学 | Klebsiella sp.with effect of promoting drought resistance of plants and application thereof |
| CN106962115A (en) * | 2017-04-05 | 2017-07-21 | 周兴虎 | A kind of method for improving willow resistance against diseases |
| CN108728386A (en) * | 2018-06-20 | 2018-11-02 | 荆门市拓达科技有限公司 | A kind of preparation method of glyphosate solid-state degradation bacterial agent |
| CN112189565A (en) * | 2020-11-11 | 2021-01-08 | 广西壮族自治区农业科学院 | Method for introducing azotobacter into sugarcane tissue culture seedlings |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103911317A (en) * | 2014-03-07 | 2014-07-09 | 中国农业科学院生物技术研究所 | Bacteria capable of resisting glyphosate, and applications thereof |
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| CN104351255A (en) * | 2014-11-10 | 2015-02-18 | 云南农业大学 | Application of Klebsiella variicola SH-1 strain |
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| CN105754901A (en) * | 2016-04-13 | 2016-07-13 | 西北农林科技大学 | Klebsiella sp.with effect of promoting drought resistance of plants and application thereof |
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| CN106962115A (en) * | 2017-04-05 | 2017-07-21 | 周兴虎 | A kind of method for improving willow resistance against diseases |
| CN108728386A (en) * | 2018-06-20 | 2018-11-02 | 荆门市拓达科技有限公司 | A kind of preparation method of glyphosate solid-state degradation bacterial agent |
| CN112189565A (en) * | 2020-11-11 | 2021-01-08 | 广西壮族自治区农业科学院 | Method for introducing azotobacter into sugarcane tissue culture seedlings |
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Application publication date: 20130313 |