CN103820359A - Acquisition of bacteria for producing ACC deaminizing enzyme and promoting growth of ginseng and application thereof - Google Patents
Acquisition of bacteria for producing ACC deaminizing enzyme and promoting growth of ginseng and application thereof Download PDFInfo
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- CN103820359A CN103820359A CN201410011921.8A CN201410011921A CN103820359A CN 103820359 A CN103820359 A CN 103820359A CN 201410011921 A CN201410011921 A CN 201410011921A CN 103820359 A CN103820359 A CN 103820359A
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Abstract
The invention discloses JJ8-3 bacterial strains of pseudomonas fluorescens of ginseng endophytic bacteria, and the preservation number is CGMCC No.8239. Among plenty of ginseng endophytic bacteria, some bacterial strains which can produce ACC deaminizing enzyme are obtained through primary selection, and the bacteria for producing the ACC deaminizing enzyme and promoting the growth of ginseng are obtained through repeatedly screening. Plenty of experiments prove that the bacterial strains JJ8-3 can produce the ACC deaminizing enzyme and have strong action for promoting the growth of the ginseng at the same time, and the bacterial strains have no pathogenicity. Through identification, the bacterial strains JJ8-3 are pseudomonas fluorescens. The disadvantages of low germination rate of ginseng seeds, slow growth of the ginseng which is one of the most important traditional Chinese medicine materials in China, poor stress resistance of the ginseng and the like are solved. The finding of the bacterial strains establishes a favorable basis for promoting the growth of the ginseng and developing and utilizing bacterial manure.
Description
Technical field
The invention belongs to microbial technology field, a specifically strain produces acc deaminase ginseng growth-promoting bacterium and application thereof.
Background technology
In recent years, the generation of natural disaster and the irrational utilization of chemical fertilizer have caused great negative impact to soil and plant growth.From environment, screening has a Promoting plant growth, and the microorganism strains that improves plant stress-resistance ability and improve environment has great importance.Some that reported at present have the bacterial strain of growth-promoting function, and its main characteristic comprises produces IAA, fixed nitrogen, phosphorus decomposing, potassium decomposing, product siderophore etc.And the report of 1-amino-cyclopropane 1-carboxylic acid (ACC) desaminase and active bacterial strain research thereof is less, it is also in recent years one of more concerned plant growth-promoting bacteria characteristic.Ethene is the important plant endogenous growth regulator of a class, mainly under fruit maturation, plant senesecence or unfavourable condition, bring into play regulating effect, also claim " stress hormone ", growth, the resistance that the excessive generation of ethene can suppress elongation, the plant of plant seed root declines, withers ahead of time etc.And acc deaminase has the effect of degrading ethylene Promoting plant growth.It is reported that acc deaminase is promoting to have important regulating effect aspect saline and alkaline, arid, the heavy metal ion of Genes For Plant Tolerance and plant growth-promoting.
Plant is coerced can produce ethene.High-caliber ethene can suppress the growth of plant.The bacterium with A CC desaminase can absorb vegetable cell and be secreted into the direct precursor A CC that is used for synthesizing ethylene outside born of the same parents, by the catalysis of A CC desaminase, A CC is hydrolyzed into a mono-ketone butyric acid and N H 3, thereby the level of Stress ethylene in reduction plant materials, removes the inhibition of Stress ethylene to plant-growth.A large amount of bacteriums that studies confirm that inoculation has an A CC desaminase can promote the growth of plant, especially suffer under the adverse circumstance of biology or abiotic stress plant.Therefore, have A C C deaminase active become assessment bacterium be an important indicator for promoting growth of plants bacterium.But, in recent years, from the genome sequence of some pathogenic bacterium, have been found that A CC deaminase gene, A CC deaminase active from some pathogenic bacterium, also detected, also found that the pseudomonas (Pseudomonas bicacearum) with A CC desaminase can allow tomato stem and fruit produce illness.
Summary of the invention
The object of the invention is that ginseng seed percentage of germination is low in order to overcome, the shortcoming such as poor growth, resistance are poor, and provide
onestrain produces acc deaminase ginseng growth-promoting bacterium.
Ginseng endogenetic bacteria fluorescent pseudomonas JJ8-3 bacterial strain, deposit number is CGMCC No.8239.
The application of ginseng endogenetic bacteria fluorescent pseudomonas JJ8-3 bacterial strain aspect promotion Ginseng Growth.
The invention provides ginseng endogenetic bacteria fluorescent pseudomonas JJ8-3 bacterial strain, deposit number is CGMCC No.8239, it is in a large amount of ginseng endogenetic bacterias, obtained several strains and can produce the bacterial strain of acc deaminase through primary election, then repeated screening obtains, through a large amount of experimental results show that, bacterial strain JJ8-3 not only can produce acc deaminase, also has very strong promotion Ginseng Growth effect, no pathogenicity simultaneously, through identify, bacterial strain JJ8-3 be fluorescent pseudomonas (
pseudomonas fluorescens), solve one of of paramount importance Chinese medicinal materials of China, the shortcoming such as the rate of emergence of ginseng is low, poor growth, resistance are poor, the exploitation and the utilization thereof that are found to be ginseng growth-promoting bacterial manure of this bacterial strain are had laid a good foundation.
Accompanying drawing explanation
Fig. 1 is JJ8-3 strains A CC deaminase activity determination, wherein, becomes brown after the reaction of 1:JJ8-3 bacterial strain; 2: contrast;
Fig. 2 is other growth-promoting functional analyses of JJ8-3 bacterial strain, wherein, and 1: phosphorus decomposing characteristic, 2: fixed nitrogen potential, 3: produce body bearer capabilities;
Fig. 3 is the colonial morphology of JJ8-3 bacterial strain on NA substratum;
Fig. 4 is the generation fluorescence of JJ8-3 bacterial strain under ultraviolet;
Fig. 5 is the thalli morphology of JJ8-3 bacterial strain electricity Microscopic observation;
Fig. 6 is the Molecular Identification cluster collection of illustrative plates of JJ8-3 bacterial strain;
Fig. 7 is the growth-promoting functions that JJ8-3 bacterial strain germinates to ginseng seed;
Fig. 8 is the growth-promoting functions that JJ8-3 bacterial strain germinates to ginseng seed;
Fig. 9 is the growth-promoting functions of JJ8-3 bacterial strain to ginseng;
Figure 10 is the growth-promoting functions of JJ8-3 bacterial strain to ginseng fresh weight;
Figure 11 is the growth-promoting functions of JJ8-3 bacterial strain to ginseng dry weight.
Embodiment
embodiment 1produce the acquisition of the active endogenetic bacteria of acc deaminase ginseng (fluorescent pseudomonas).
Adopt plate dilution method separation to obtain 120 strains and derive from the ginseng endogenetic bacteria in Jilin Province, and it has been carried out to the screening of acc deaminase active bacterial strain.
Screening is divided into primary dcreening operation and multiple sieve, and primary dcreening operation adopts liquid culture method to screen 120 strain bacteriums, bacterial strain to be measured is inoculated into respectively in liquid LB substratum, and 28 ℃, 160 r/min incubated overnight.Get 0.2 mL substratum and be transferred in 8mL DF salt culture medium, under same culture conditions, cultivate 12 h.And then get the 0.2mL substratum 8mL ADF liquid nutrient medium of again transferring.And with containing the ADF substratum of ACC not in contrast.Cultivate 24 ~ 48h and survey its 600 nm place light absorption value, repeat for three times, the positive bacterial strain significantly increasing than contrast light absorption value.Result screening obtains screening 8 strain positive strains.(DF salt culture medium: KH
2pO
44.0 g, Na
2hPO
46.0 g, MgSO
47H
2o 0. 2 g, glucose 2.0 g, gluconic acid 2.0 g, citric acid 2. 0 g, (NH
4)
2sO
42. 0 g, deionized water 1000mL, pH 7.2; ADF substratum: ACC is dissolved in after the ultrapure water of sterilizing, through the disposable sterilized strainer filtration sterilization of 0.22 μ m, is added to and does not contain (NH
4)
2sO
4dF salt culture medium in, final concentration is 3.0 mmol/L.）
The bacterial strain that multiple sieve adopts α-one butyric acid method to obtain primary dcreening operation screens: 1. inoculation is in LB liquid nutrient medium, 28 ℃, 160 r/min incubated overnight, nutrient solution after the centrifugal 5min of 8000 r/min by thalline Eddy diffusion in 7.5 mL ADF liquid nutrient mediums, 28 ℃, 160 r/min continue to cultivate 16 h.Take out nutrient solution 8000 r/min centrifugal, abandon supernatant, collect thalline.Thalline is suspended in ultrasonication after the Tris-HCl damping fluid of 0.1 mol/L again, obtains crude enzyme liquid and preserves; 2. the drafting of α-one butyric acid typical curve, draws respectively α-one butyric acid standard model 200 μ L and adds in sterile test tube, separately adds 1.6 mL HCl(0.56 mol/L) and 300 μ L 2,4 dinitrophenyl hydrazines (0.2%), 30 ℃ of water-bath 30 min.After taking-up, adding 2 mL NaOH(2mol/L) solution mixes, and 540nm measures light absorption value.3. get two test tubes, add respectively 200 μ L crude enzyme liquids.Wherein in a test tube, add 0.5mmol ACC 20 μ L to mix, after 30 ℃ of water-bath 15min, add 1mL 0.56 mol/L HCl termination reaction, the centrifugal 5min of 12000r/min, get supernatant liquor 1mL, add 2 of 800 μ L 0.56 mol/L and 300 μ L 0.2%, 4-dinitrophenylhydrazine, 30 ℃ of water-bath 30min, add 2mL 2mol/L NaOH solution to mix after taking-up.It is control tube that another test tube does not add 0.5mmol ACC, measures light absorption value at 540nm place.Sieve again experimental result and show, JJ8-3 bacterial strain has acc deaminase activity (Fig. 1), and its enzymic activity is α-one butyric acid 6.702 μ mol/mgh.
embodiment 2: the mensuration of other growth-promoting indexs of active bacterial strain JJ8-3
1, separate the screening of inorganic phosphorus ability: primary dcreening operation adopts PKO plate method, and JJ8-3 bacterial strain is cultivated after 12h on NA substratum, plays bacterium dish and is inoculated in PKO solid medium, observes phosphorus decomposing circle size after 28 ℃ of constant temperature culture 3d.Multiple sieve adopts molybdenum antimony scandium colorimetry, and JJ8-3 punching is inoculated in to 50mL LB liquid nutrient medium, and 160 r/min cultivate 12h.Get 1 mL and be inoculated in the 150 mL triangular flasks that 50 mLPKO liquid nutrient mediums are housed, get nutrient solution 5mL after 3 d, after the centrifugal 5min of 8000 r/min, get supernatant, molybdenum antimony scandium colorimetry is measured titanium pigment content.Result demonstration, on PKO flat board, JJ8-3 bacterial strain has obvious phosphorus decomposing circle (Fig. 2); It separates phosphorus content quantitative assay, can reach 380 μ g/mL.
2, the mensuration of nitrogen fixing capacity: on Ashby solid medium, 30 ℃ of constant temperature culture 2d, observe bacterial strain growing way, found that bacterial strain JJ8-3 is without grow fine under nitrogen condition (Fig. 2) by bacterial strain streak inoculation.
3, produce the mensuration of siderophore ability: primary dcreening operation adopts plate method, and JJ8-3 bacterial strain is cultivated after 12h on NA substratum, plays bacterium dish and is inoculated in resazurin solid medium, after 28 ℃ of constant temperature culture 2d, observes periphery of bacterial colonies colour-change.Multiple screen method is colorimetry, encircle fresh lawn in MKB nutrient solution with transfering loop picking one, 28 ℃, 160r/min shaking table are got the centrifugal 10min of bacterium liquid 10000r/min after cultivating 2d, get supernatant 3ml in test tube, add 3ml CAS to detect liquid and mix rear 20 ℃ of reaction 1h, measure light absorption value As at 630nm place; Separately get 3ml CAS and detect the MKB liquid nutrient medium supernatant liquor that liquid and 3ml do not connect bacterium and mix, survey equally light absorption value Ar.According to formula ((Ar-As)/Ar) × 100) calculating siderophore generation.Result demonstration, JJ8-3 bacterial strain has the siderophore activity of generation (Fig. 2), and its activity is up to 91.8%.
embodiment 3: the evaluation of bacterial strain JJ8-3
The streak inoculation of JJ8-3 bacterial strain, on NA substratum, is cultivated after 12 ~ 24h to observed and recorded list colonial morphology for 28 ℃.JJ8-3 is the opaque circular bacterium colony of light green on NA substratum, and edge complete sub-translucent (Fig. 3), Gram-negative, without mobility, observes under ultraviolet and produces fluorescence (Fig. 4).
Be taken at the JJ8-3 bacterial strain bacterium liquid of cultivating 24h in LB liquid nutrient medium, centrifugal repeatedly rinse with sterilized water after, be suspended in and in appropriate sterilized water, carry out negative staining and prepare sample, clamp with the copper mesh of film and dip micro-bacterium liquid with tweezers, leave standstill 2 min, blot residual solution from edge with filter paper; And then clamp with tweezers the copper mesh that carries disease germs and immerse rinsing in the beaker that fills distilled water, then blot liquid with filter paper, then with 2% phospho-wolframic acid negative staining 5 min, be shaft-like with transmission electron microscope observing discovery thalline after dry, there is extreme growth flagellum (Fig. 5).
Physio-biochemical characteristics with reference to the part substratum (table 1) of recommending in " common bacteria system identification handbook " and " the outstanding system identification handbook of uncle " and method mensuration JJ8-3 bacterial strain are found, JJ8-3 bacterial strain catalase, oxydase reaction are positive, and V-P reaction is negative; Can utilize citric acid, can not make nitrate reduction, Starch Hydrolysis, but can make fat hydrolysis, gelatine liquefication, litmus milk reduces but does not peptonize, and is 7% to the tolerance of NaCl; Can utilize glucose, N.F,USP MANNITOL and semi-lactosi.
Obtain sequence in bacterial strain 16S rDNA sequence and GenBank database and carry out BLAST analyses and comparison, the accession number of acquisition is KF727589, and constructing system evolutionary tree sees Fig. 6.JJ8-3 bacterial strain with
pseudomonas fluorescensgather in same branch, its similarity reaches 99.46%.By morphological specificity, cultural characteristic, physiological and biochemical property and molecular biology identification result, JJ8-3 bacterial strain be fluorescent pseudomonas (
pseudomonas fluorescens).
Fluorescent pseudomonas JJ8-3 bacterial strain, on September 24th, 2013, deliver to China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) preservation, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, deposit number is CGMCC No.8239.
embodiment 4: the growth-promoting effect of bacterial strain JJ8-3 to ginseng
1. seed germination experiment
After bacterial strain JJ8-3 inoculation LB nutrient solution shaking table incubated overnight, the centrifugal 5min of 8000 rpm collects thalline.After washing 2 times with sterilized water, be resuspended in sterilized water, blood counting chamber is measured concentration and is diluted to concentration is 10
9the bacterial suspension of cfu/mL.Select the ginseng seed of after-ripening to be immersed in 3h in bacteria suspension, take out and dry after surface liquid, be placed in the culture dish that aseptic moistening sand is housed, 15 seeds of every ware, are 1 repetition, repeat for 6 times, place vernalization 10 ~ 15d for 18 ~ 23 ℃ in dark place, take the seed of sterilized water processing as contrast.Result shows (table 2, Fig. 7, Fig. 8), and the long mean value of ginseng seed bud of JJ8-3 bacterial strain processing is 20.5 mm, is greater than clear water control treatment (12.2 mm), and bud is long increases by 68.03%, shows that JJ8-3 bacterial strain has promoter action to ginseng seed germinating growth.
2. seed sprouting test
The ginseng seed of processing through bacterial strain JJ8-3 bacteria suspension is implanted in sandy soil culturing pot, and planting depth 2 cm, beat vola water, and 10 seeds of every alms bowl repeat, contrast 18 ℃ ~ 23 ℃ growths, observed and recorded seedling-growing time after 7 days with the seed of sterilized water processing for 3 times.Test-results shows, when the seeding ratio that JJ8-3 processes reaches 70%, needs 10 days, 5 days in advance compared with the control.
3. the growth-promoting effect of pair ginseng
Test and on October 20th, 2012,2 years of uniform size stranger's seedlings are planted in the consistent people's ginseng bed of soil fertility, planting depth 5cm.On June 5th, 2013, ginseng is emerged and is opened up completely after leaf with 10
8the JJ8-3 bacteria suspension of cfu/mL carries out root irrigation, every repetition 10 strain ginseng-leafs, and pouring 200mL bacteria suspension, the water of contrast pouring same volume, repeats for 4 times, within every 10 days, waters once totally 3 times.After on October 25th, 2013, ginseng overground part cauline leaf came off, trip out ginseng, 10 strains are that unit measures root fresh weight and dry weight.
Detected result shows, to ginseng, growth has obvious promoter action to JJ8-3 bacterial strain, and through the ginseng of JJ8-3 bacterial strain processing compared with the control, fresh weight increases by 31.72%, and dry weight increases by 37.94%, and phase statistics is in table 2, Fig. 9, Figure 10, Figure 11.
Claims (2)
1. ginseng endogenetic bacteria fluorescent pseudomonas JJ8-3 bacterial strain, deposit number is CGMCC No.8239.
2. the application of ginseng endogenetic bacteria fluorescent pseudomonas JJ8-3 bacterial strain claimed in claim 1 aspect promotion Ginseng Growth.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106318872A (en) * | 2016-11-23 | 2017-01-11 | 吉林农业大学 | Ginseng growth promoting bacteria JI39-2 and application thereof |
| CN106434490A (en) * | 2016-11-23 | 2017-02-22 | 吉林农业大学 | Ginseng bacterium TY15-2 with effects of disease prevention and growth promotion and application thereof |
| CN109880757A (en) * | 2019-03-04 | 2019-06-14 | 西北大学 | One plant of hydrogen-oxidizing bacterium and its separation method and application with itself nitrogen fixing capacity |
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| CN102250808A (en) * | 2011-07-15 | 2011-11-23 | 中国农业科学院农业资源与农业区划研究所 | Endophytic azotobacter of wheat producing ACC (1-aminocyclopropane-1-carboxylate) deaminase and application thereof |
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| CN102250808A (en) * | 2011-07-15 | 2011-11-23 | 中国农业科学院农业资源与农业区划研究所 | Endophytic azotobacter of wheat producing ACC (1-aminocyclopropane-1-carboxylate) deaminase and application thereof |
| CN102787090A (en) * | 2012-08-07 | 2012-11-21 | 哈尔滨师范大学 | Alfalfa root growth-promoting rhizobacteria MJM-5 and application of alfalfa root growth-promoting rhizobacteria MJM-5 |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106318872A (en) * | 2016-11-23 | 2017-01-11 | 吉林农业大学 | Ginseng growth promoting bacteria JI39-2 and application thereof |
| CN106434490A (en) * | 2016-11-23 | 2017-02-22 | 吉林农业大学 | Ginseng bacterium TY15-2 with effects of disease prevention and growth promotion and application thereof |
| CN106434490B (en) * | 2016-11-23 | 2019-04-02 | 吉林农业大学 | Ginseng disease prevention growth-promoting bacterium TY15-2 and its application |
| CN106318872B (en) * | 2016-11-23 | 2019-05-07 | 吉林农业大学 | Ginseng growth-promoting bacterium JI39-2 and its application |
| CN109880757A (en) * | 2019-03-04 | 2019-06-14 | 西北大学 | One plant of hydrogen-oxidizing bacterium and its separation method and application with itself nitrogen fixing capacity |
| CN109880757B (en) * | 2019-03-04 | 2022-05-17 | 西北大学 | Hydrogen hydroxide bacterium with self nitrogen fixation capacity and separation method and application thereof |
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