CN102816720B - ACC-deaminase-producing serratia marcescens LJL-11 and application thereof - Google Patents

ACC-deaminase-producing serratia marcescens LJL-11 and application thereof Download PDF

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CN102816720B
CN102816720B CN201210284868.XA CN201210284868A CN102816720B CN 102816720 B CN102816720 B CN 102816720B CN 201210284868 A CN201210284868 A CN 201210284868A CN 102816720 B CN102816720 B CN 102816720B
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serratia marcescens
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郭长虹
刘佳莉
郇菲菲
束永俊
陶楠
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Harbin Normal University
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Abstract

The invention discloses an ACC-deaminase-producing serratia marcescens LJL-11 and an application thereof. The categorical name of the serratia marcescens is serratia marcescens LJL-11. The serratia marcescens is collected in China General Microbiological Culture Collection Center (CGMCC) with a collection number of CGMCC NO. 6290. The stain can grow with 1-amino cyclopropane-1-carboxylic acid (ACC) as a sole nitrogen source, and can decompose the sole nitrogen source. ACC deaminase activity of the stain reaches a maximal value of 38.52mumol alpha-KA.(mg Pr.h)<-1>. With the increasing of L-Trp concentration, IAA synthesis amount of the strain is increased. The strain can also synthesize siderophore. The strain can be used for improving plant stress resistance in stress environments of salinity, drought, metal, organic pollution, and the like, and assists in promoting plant growth. The strain has a promotion effect upon the growth of oat in petroleum contaminated saline-alkali soil.

Description

A kind of serratia marcescens LJL-11 and application thereof of producing acc deaminase
Technical field
The invention belongs to plant growth-promoting bacteria technical field, relate to a kind of serratia marcescens LJL-11 and application thereof of producing acc deaminase.
Background technology
In recent years, along with Economic development and global environmental change, salting of soil, heavy metal contamination, arid and Organic pollutants are more and more serious, and these adverse environmental factors have caused the underproduction of farm crop.How effectively improving the anti-adversity ability of plant and the output of increase farm crop has become the problem of extensive concern.
Environment-stress can cause a large amount of accumulation of Stress ethylene in plant materials, and that excessive ethene can cause plant-growth to be obstructed is even dead.1-amino-cyclopropane-1-carboxylic acid (1-aminocyclopropane-1-carbo xylate, ACC) is the synthetic precursor of ethene in object.A lot of research is found, various plants rhizosphere growth-promoting endophytic bacteria (plant growth-promoting rhizobacteria, PGPR) can secrete and a kind ofly can suppress polysaccharase in the synthetic born of the same parents of ethene in plant materials, acc deaminase (ACC deaminase), ACC can be decomposed into α-batanone acid and ammonia, can reduce the generation of Stress ethylene, thereby improve the adaptive faculty of plant under environment stress.Therefore, urgently develop a kind of harm that can effectively alleviate environment stress and plant rhizosphere growth-promoting bacterium that can Promoting plant growth.
Summary of the invention
The problem that the present invention solves is to provide a kind of serratia marcescens LJL-11 and application thereof of producing acc deaminase, and this serratia marcescens is a kind of new plant growth-promoting bacteria, is expected to play a significant role in the phytoremediation of the salt affected soil of petroleum pollution.
The present invention is achieved through the following technical solutions:
A kind of serratia marcescens LJL-11 that produces acc deaminase, its Classification And Nomenclature is serratia marcescens LJL-11(Serratia marcescens), be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), preserving number is CGMCC NO.6290.
This bacterium can be synthesized acc deaminase in born of the same parents, under aerobic condition, ACC is grown as only nitrogen source, simultaneously by its decomposition.
Further, this bacterium can synthesis of indole acetic acid.
And this bacterium can be synthesized and had a liking for iron element.
The application of this bacterium comprises:
Described serratia marcescens LJL-11 is in the application of promoting growth of plants.
The described application preparing preparation or the raising stress resistance of plant of promoting growth of plants.
Described preparation is for suppressing the preparation of ACC generation, synthetic preparation, short one or more that have a liking in the synthetic preparation of iron element of short indolylacetic acid.
The application of the serratia marcescens LJL-11 of described product acc deaminase in the phytoremediation of the soil of petroleum pollution.
The described application that improves the preparation of the resistance in the salt affected soil of plant in petroleum pollution in preparation.
The serratia marcescens LJL-11 of described product acc deaminase is coercing under environment and is synthesizing to improve the application in anti-adversity ability by suppressing ethene in body plant.
Compared with prior art, the present invention has following useful technique effect:
The serratia marcescens LJL-11 of product acc deaminase provided by the invention, to using ACC, as the substratum in unique N source, the microorganism in the Rhizosphere Soil of growing plants the saline-alkali soil from petroleum pollution is carried out to screening and separating, what obtain can using the microorganism of ACC as unique N source growth, detected result shows that this bacterium is a kind of new serratia marcescens that can synthesize acc deaminase, and Classification And Nomenclature is Serratia marcescens.
The serratia marcescens LJL-11 of product acc deaminase provided by the invention, due to its synthetic acc deaminase, allly can suppress the synthetic of ACC in plant materials, and then reduce because environment-stress causes a large amount of accumulation of ethene in plant materials, thereby improve its resistance.
The serratia marcescens LJL-11 of product acc deaminase provided by the invention, can also synthesis of indole acetic acid and have a liking for iron element, thereby can help plant that indolylacetic acid is provided and promote the absorption of ferro element, plays the effect of promoting growth of plants.
The serratia marcescens LJL-11 of product acc deaminase provided by the invention, growth to oat in the saline-alkali soil of petroleum pollution has promoter action, the growth promoting function that comprises plant height, strain fresh weight, root length and root fresh weight: compared with the control, plant height increases by 42.57%, the strain of plant fresh weight/5 increases by 9.83%, root is long increases by 10.22%, and the strain of root fresh weight/5 increases by 3.45%.This bacterial strain can be coerced the anti-adversity ability that improves plant under environment, Promoting plant growth at saline and alkaline, arid, metal, Organic pollutants etc.
Preservation explanation
Serratia marcescens of the present invention (Serratia marcescens) LJL-11 has carried out following preservation:
The preservation time: on June 26th, 2012, preservation place: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica; Depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center, CGMCC; Preserving number is CGMCC NO.6290.
Accompanying drawing explanation
Fig. 1 is the upgrowth situation of serratia marcescens (Serratia marcescens) LJL-11 on ADF substratum.
Fig. 2 is the variation of serratia marcescens (Serratia marcescens) LJL-11 IAA resultant quantity in different concns L-Trp.
Fig. 3 is that serratia marcescens (Serratia marcescens) LJL-11 forms safran haloing on chromium alcohol CAS difficult to understand solid medium.
Fig. 4 is serratia marcescens (Serratia marcescens) LJL-11 to oat growth-promoting pot test effect in the time of 3 weeks.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in further detail, and the explanation of the invention is not limited.
The experimental technique using in following embodiment if no special instructions, is ordinary method.
In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.
Separated and the evaluation of embodiment 1, serratia marcescens (Serratia marcescens) LJL-11
1, the separation of serratia marcescens (Serratia marcescens) LJL-11
The separation of serratia marcescens (Serratia marcescens) LJL-11 comprises sampling, screening and two steps of purifying, and concrete grammar is as follows:
1.1, sampling
The soil of screening plant growth-promoting rhizobacteria is taken near the Rhizosphere Soil of the saline and alkaline geophilous oat in oil recovery factory, Daqing, Heilongjiang Province, and Rhizosphere Soil is packed in sterilized collecting bottle and takes back laboratory, puts into 4 ℃ of Refrigerator stores standby.
1.2, screening and purifying
(1) get 1g Rhizosphere sampling in 50mL PAF substratum, 28 ℃ of shaking culture 24h.In this PAF substratum, contain peptone, casein hydrolysate, MgSO 4, K 2hPO 4glycerine, specifically with reference to Penrose D M, Glick B R.Methods for isolating and characterizing ACC deaminase-containing plant growth-promoting rhizobacteria[J] .Physiologia Plantarum, 2003,118 (1): 10-15 prepares.
(2) get the PAF nutrient solution (suspension) after 1mL above-mentioned (1) concussion, in 50mL PAF substratum, 28 ℃ of shaking culture 24h.
(3) get the PAF nutrient solution that 1mL above-mentioned (2) obtains, in 50mL DF salt nutrient solution, 28 ℃ of shaking culture 24h.This DF salt nutrient solution contains KH 2pO 4, Na 2hPO 4, MgSO 4, FeSO 4, glucose, gluconic acid, citric acid, (NH 4) 2sO 4, H 3bO 3, MnSO 4, ZnSO 4, CuSO 4, MoO 3.
(4) get the DF salt nutrient solution that 1mL above-mentioned (3) obtains, in 50mL, do not contain (NH 4) 2sO 4, but in the DF salt nutrient solution that contains 3mM 1-amino-cyclopropane-1-carboxylic acid (ACC), (using ACC as unique N source), 28 ℃ of shaking culture 48h.
(5) get the nutrient solution that 1mL above-mentioned (4) obtains, coat on the DF salt nutrient agar containing 3mM ACC 28 ℃ of cultivations.
(6) get the bacterium colony of growing on above-mentioned (5) substratum, the purifying of ruling on ADF substratum, obtains single bacterium colony.
Like this through usining ACC as the substratum in unique N source to utilizing its bacterium as the growth of N source to screen in Rhizosphere Soil, thereby obtained single bacterium colony.
2, the evaluation of serratia marcescens (Serratia marcescens) LJL-11
The pure culture bacterial strain that above-mentioned separation and purification is obtained carries out a series of Physiology and biochemistry evaluations, and carries out DNA extraction, the amplification of 16S rDNA and order-checking.
2.1 these bacterial strains form red, opaque, the irregular bacterium colony in edge on ADF substratum, as shown in Figure 1.
2.2 utilize primers F 8 and the R1541 16S rDNA that increases, and primer sequence is as follows:
F8：5'AGAGTTTGATCCTGGCTCAG3′，
F 1541：5'AAGGAGGTGATCCAGCCGCA3′。
Pcr amplification condition is: 94 ℃ of 3min; 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 90s, 30 circulations; 72 ℃ of 10min.
Pcr amplification product is checked order, and sequencing result is as shown in SEQ.ID.NO.1.
By strain called after serratia marcescens (Serratia marcescens) LJL-11 in the pure culture bacterial strain of above-mentioned acquisition, and by its preservation, preserving number is CGMCC NO.6290.
The acc deaminase determination of activity of embodiment 2, CGMCC NO.6290
Strains tested CGMCC NO.6289 is incubated overnight in TSB nutrient solution (NaCl 5.0g, glucose 2.5g, K2HPO42.5g, distilled water 1000mL, pH 7.5 for Tryptones 17.0g, soya peptone 3.0g), 4 ℃ of centrifugal collection thalline.DF nutrient solution washing three times for somatic cells, Eddy diffusion is in ADF nutrient solution, and 28 ℃ of shaking culture of room temperature are after 2 days, and 4 ℃ of centrifugal collection thalline, use 0.1molL -1tris-HCl damping fluid (pH=7.6) washs centrifugal three times, is resuspended to 600 μ L 0.1molL -1in Tris-HCl damping fluid (pH=8.0), add 30 μ L toluene rapid vibration 30s with smudge cells, transferase 12 00 μ L contains the cell extract of toluene to 1.5mL centrifuge tube, adds 20 μ L 0.5molL -1aCC mixes, and does the blank test of not adding ACC simultaneously, cultivates 15min for 30 ℃.Add 1mL 0.56molL -1hCl, the centrifugal 5min of 16000g, gets 1mL suspension to test tube, adds 800 μ L 0.56molL -1hCl and 300 μ L 2,4 dinitrophenyl hydrazines, hatch 30min for 30 ℃, adds 2mL 2molL -1naOH, 540nm wavelength is surveyed absorbancy.Take 1mL concentration as 0,0.1,0.3,0.7,1 and 2 μ molL -1α-batanone acid be reference liquid, survey light absorption value under 540nm wavelength.
Protein determination adopts Xylene Brilliant Cyanine G method to measure.Take 1mL concentration as 0,20,40,60,80 and 100 μ gL -1bovine serum albumin solution be standardized solution, add 5mL Coomassie brilliant blue G250 staining fluid, reaction is measured light absorption value under 595nm after 5min.
It is μ mol α-KA (mgPrh) that the activity of acc deaminase be take the scale Shi， unit of every milligram of albumen formation per hour α-batanone acid in surveying enzyme system -1.Blank rear calculating of sample contrast all deducted in enzyme assay, repeats 3 times.
Adopting α-pyruvic acid standard substance is standard production standard curve, and typical curve equation (equation first) is as follows: y=3.6928x-0.035(R 2=0.9879), wherein x represents OD 540nmnumerical value, y represents α-batanone acid content (μ mol).Adopt Xylene Brilliant Cyanine G method to detect the total protein content of cell extract.The employing bovine serum albumin of take is standard substance production standard curve, and typical curve equation (equation second) is as follows: y=205.73x-1.3251(R 2=0.9846), wherein x represents OD 595nmnumerical value, y represents protein content (mg).
According to production standard curve and detected result, can obtain the activity of acc deaminase activity.Detected result shows to have acc deaminase activity in the cytoclasis thing of CGMCC NO.6289, and enzymic activity reaches as high as 38.52 μ mol α-KA (mg Prh) -1, this shows that CGMCC NO.6290 can synthesize acc deaminase in born of the same parents.
The IAA resultant quantity of embodiment 3, CGMCC NO.6290 is measured
CGMCC NO.6290 is first cultivated 2 days in DF nutrient solution, then the DF nutrient solution that trace proceeds to interpolation different concns tryptophane (L-Trp) is (containing 0,50,100,200 and 500 μ g L-TrpmL -1) middle cultivation 2 days, the sampling survey bacterium liquid OD of continuing 600, under all the other nutrient solution room temperatures, 8000g is centrifugal, gets 500 μ L supernatant liquors, adds 2mL Salkowski reagent (containing 150mL H 2sO 4, 250mL ddH 2o and 7.5mL 0.5molL -1feC1 3), after dark lower incubated at room temperature 20min, at 535nm place survey light absorption value (OD535), aseptic culture medium is the same to be done identical processing and returns to zero in contrast.Take concentration as 0,0.01,0.05,0.25,0.5mgmL -1iAA reference liquid with method, do typical curve, typical curve equation is as follows: y=0.1701x-0.0237(R 2=0.9855), wherein x represents OD 535nmnumerical value, y represents IAA concentration (μ gmL -1).
According to production standard curve and detected result, can obtain the IAA resultant quantity of CGMCC NO.6290; And detected result also shows, serratia marcescens (Serratia marcescens) LJL-11 can utilize tryptophane to produce indolylacetic acid; As shown in Figure 2, and along with the increase of L-Trp concentration, the amount of the IAA that serratia marcescens (Serratia marcescens) LJL-11 is synthetic is constant.
CGMCC NO.6290 be take tryptophane (L-Trp) as the synthetic plant growth hormones indolylacetic acid (IAA) of precursor, the surface that can be adsorbed on seed and root is utilized by plant, simultaneously also can with IAA acting in conjunction stimulating plant Growth of Cells and the propagation of plant endogenesis, can promote growing and effectively absorbing moisture and the nutrient in soil of root system of plant, other vital movements of plant be regulated simultaneously.
The iron element resultant quantity of having a liking for of embodiment 4, CGMCC NO.6290 is measured
Method (Schwyn B with reference to Schwyn and Neilands, Neilands J B.Universal chemical assay for the detection and determination of siderophores[J] .Analytical Biochemistry, 1987,160:47-56.), serratia marcescens LJL-11 is had a liking for to the synthetic qualitative experiment of iron element.Serratia marcescens (Serratia marcescens) LJL-11 is connected to chromium alcohol CAS(chrome difficult to understand azurol S) on solid medium, cultivate 48h for 28 ℃, observe the colour-change of periphery of bacterial colonies, there is orange chromosphere to produce to produce and have a liking for iron element.
As shown in Figure 3, serratia marcescens (Serratia marcescens) LJL-11 periphery of bacterial colonies on CAS agar plate has safran haloing to form to detected result, has produced and has had a liking for iron element.
With reference to the method for Zhao Xiang etc., measure and have a liking for iron element.Serratia marcescens (Serratia marcescens) LJL-11 is inoculated in MKB substratum to 28 ℃ of 180rmin -1shaking table is cultivated 48h; Bacterium liquid is centrifugal, gets supernatant liquor, with the volume ratio of 1:1, adds CAS to detect liquid, fully mixes.After static 1h, 630nm place surveys light absorption value (A), and the Ar that deionized water records with the mixing of CAS detection liquid equal-volume, for contrasting, has a liking for the relative content of iron element in A/Ar representative sample, and this value is lower, shows to have a liking for iron cellulose content higher.A/Ar<0.5, can think that product is had a liking for iron element ability higher.Result shows, the A/Ar value of serratia marcescens (Serratia marcescens) LJL-11 is 0.71, and close to 0.5, it is described, and synthetic to have a liking for iron element ability higher.Like this CGMCC NO.6290 can by produce and secrete iron is had to a high-affinity have a liking for iron element, dissolve also in conjunction with the ferro element in soil for vegetable cell utilization, increase iron nutrition, Promoting plant growth.
Embodiment 5, the CGMCC NO.6290 growgh promoting effects to oat under petroleum-polluted saline alkali soil environment
For CGMCC NO.6290, the salt affected soil of oat Biological control is taken to Daqing, Heilongjiang Province, 5 sample prescriptions are set during collected specimens in each region, sample area is 1 * 1m 2, collect topsoil (0 ~ 20cm), after mixing with soil, pack in the clean freshness protection package of previously prepd, rapidly soil sample is taken back to laboratory, soil, through pulverizing, mixes, the preservation of sieving after air-dry.Supply the physico-chemical property of examination soil in Table 1.
Table 1 is for examination soil physico-chemical property
Figure BDA00001997806700081
CGMCC NO.6290 is inoculated in TSB liquid nutrient medium, shaking culture at 28 ℃, and 4 ℃ of centrifugal collection thalline, are resuspended in sterilized water after washing centrifugal 2 times with sterilized water, make the final concentration of viable count in sterilized water reach 10 9cFU/mL.
Oat seed, with washing with sterilized water after 0.5% (V/V) clorox surface sterilization 10min, is then used to the nutrient solution seed soaking 2h of CGMCC NO.6290, make it be attached to seed-coat.
CGMCC NO.6290 is processed to the oat seed of 2h and the oat seed (CK) of processing without CGMCC NO.6290, evenly plant in being mixed with the saline-alkali soil of oil (petroleum concentration is 4.5g/kg dry ground) respectively, every basin 10 strains, repeat 3 times, in culturing room, (25 ℃) are cultivated, and water every other day.After 3 weeks, weigh individual plant high, root length and 5 strain plant fresh weights and root fresh weight.
Outward appearance contrasts as shown in Figure 4, can see, after LJL-11 processes, the growing way of oat is significantly better than untreated contrast.
High, the strain fresh weight of the individual plant that weighs, root is long and the result of the contrast number of root fresh weight is as shown in table 2.
Result shows that serratia marcescens (Serratia marcescens) LJL-11 viable bacteria has significant promoter action to the plant height of oat.The oat that mensuration is processed through serratia marcescens (Serratia marcescens) LJL-11, compared with the control, plant height increases by 42.57%, and the strain of plant fresh weight/5 increases by 9.83%, and root is long increases by 10.22%, and the strain of root fresh weight/5 increases by 3.45%.The results are shown in Table 2.
Table 2 serratia marcescens LJL-11 is the growth-promoting effect to oat in petroleum pollution saline-alkali soil
Individual plant high (cm) Plant fresh weight (g)/5 strain Root long (cm) Root fresh weight (g)/5 strain
CK 16.16±1.58b 0.61±0.07a 11.74±2.19b 0.29±0.09a
LJL-11 23.04±0.29a 0.67±0.08a 12.94±0.61a 0.30±0.04a
Note: the significant difference of letter representation in 0.05 level in table
To sum up detect and show, its synthetic acc deaminase of CGMCC NO.6290, thus can suppress the synthetic of the interior ACC of plant materials, and then reduce because environment-stress causes a large amount of accumulation of ethene in plant materials, thereby improve its resistance; But also can synthesis of indole acetic acid and have a liking for iron element, thereby can help plant that indolylacetic acid is provided and promote the absorption of ferro element, play the effect of promoting growth of plants.Therefore, the general character of the promoting growth of plants having due to above-mentioned effect, so this bacterium can also be applied in the middle of Plant growth-promoting effect as a kind of bacterium of growth-promoting widely.
Further, CGMCC NO.6290 shows the growgh promoting effects of oat under the petroleum-polluted saline alkali soil environment to petroleum pollution, this bacterium can be by improving plant in the application of coercing the anti-adversity ability under environment, thereby be applied to the phytoremediation of the soil of petroleum pollution; And improve that to coerce be mainly to coerce the accumulation of ACC in plant under environment by degraded, so coerce environment, comprise that saline and alkaline, arid, metal, Organic pollutants etc. coerce environment.
Figure IDA00001997807700011
Figure IDA00001997807700021

Claims (7)

1. a serratia marcescens LJL-11 who produces acc deaminase, is characterized in that, its Classification And Nomenclature be serratia marcescens ( serratia marcescens), being preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), preserving number is CGMCC NO.6290;
This bacterium can be synthesized acc deaminase in born of the same parents, under aerobic condition, ACC is grown as only nitrogen source, simultaneously by its decomposition;
This bacterium can synthesis of indole acetic acid;
This bacterium can be synthesized and had a liking for iron element.
2. the serratia marcescens LJL-11 of product acc deaminase claimed in claim 1 is in the application of promoting growth of plants.
3. application as claimed in claim 2, is characterized in that, in the application of preparing the preparation of promoting growth of plants.
4. application as claimed in claim 3, is characterized in that, described preparation is for suppressing the preparation of ACC generation, synthetic preparation, short one or more that have a liking in the synthetic preparation of iron element of short indolylacetic acid.
5. the application of the serratia marcescens LJL-11 of product acc deaminase claimed in claim 1 in the phytoremediation of the soil of petroleum pollution.
6. application as claimed in claim 5, is characterized in that, improves the application of the preparation of the resistance in the salt affected soil of plant in petroleum pollution in preparation.
7. the serratia marcescens LJL-11 of product acc deaminase claimed in claim 1 is coercing under environment and is synthesizing to improve the application in anti-adversity ability by suppressing ethene in body plant.
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魏素娜.小麦根际具有ACC脱氨酶活性细菌菌株的分离、鉴定及接种效应的研究.《中国优秀硕士学位论文全文数据库 农业科技辑》.2012,第D047-91页.

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