CN1900269A - Gene engineering bacteria for producing crown bacterin and its construction method - Google Patents

Abstract

The present invention relates to gene engineering bacterial strain, and is especially one kind of soybean disease causing variety (P.syringae pv.Glycinea) of pseudomonas syringae for producing crown bacterin and its construction process. The gene engineering pseudomonas strain is prepared through mutagenizing initial pseudomonas strain with nitrosoguanidine for genetic mutation, so as to screen out pseudomonas strain with high crown bacterin yield. Experiment proves that the gene engineering pseudomonas strain of the present invention may be used in industrial production of crown bacterin through fermentation at 18deg.c to reach the yield of 84.7-112 mg/L.

Description

A kind of genetic engineering bacterium and construction process thereof that is used to produce psendomonas syringae

Technical field

The invention belongs to microbial technology field, specifically, relate to a kind of genetic engineering bacterium and construction process thereof that is used to produce psendomonas syringae.

Background technology

Psendomonas syringae (Coronatine) C ₁₈H ₂₅NO ₄Molecular weight is 319 (Ichihara etal, 1977), by two components of hat bacterium acid (coronafacic acid) that contain an amino acid whose hat alkanoic acid (coronamic acid) and a polyketone structure with amido bond link (Mitchell etal, 1994).Initial document is reported for work, and it is a kind of plant poison.But discover that recently psendomonas syringae and plant growth regulating substance dormin, jasmonic have similar function, and activity is both thousands of times of back, can be used as the surrogate of back both (comparatively expensive, very difficult popularization is opened productions in).And psendomonas syringae belongs to the pure natural product, on producing, use can not pollute to environment, and be a kind of environmentally friendly biological regulator.

Psendomonas syringae is synthetic two kinds of methods, and a kind of is chemical synthesis (the former people of obtaining in city etc., 1998), but expense is higher at present, can not suitability for industrialized production; Another kind is a biological synthesis process, i.e. microbe fermentation method.The initial former reed people in city (1998) just win psendomonas syringae 60mg with fermentation method from the 250L fermented liquid.Some investigators adopt biotechnology means such as genetically engineered that its output is brought up to 20-40mg/L (Bender et.al, 1996 subsequently; Ullrich, 2000).But production cost is still than higher, during the appropriate scale of agriculture that is difficult to have high input is for the moment produced.The greatest factor that limits its industrialization is exactly the strain excellent that lacks high yield, thereby breeding high-yield psendomonas syringae engineering bacteria is significant.

Summary of the invention

The object of the present invention is to provide a kind of engineering strain that is used to produce psendomonas syringae.

Another object of the present invention is to provide the construction process of this engineering strain.

Further purpose of the present invention is to provide the application of this genetic engineering bacterium.

The inventor has obtained a kind of pathogenic mutation (Pseudomonas syringae pv.glycinea) bacterial strain of pseudomonas syringae soybean of high yield psendomonas syringae, thereby has finished the present invention through further investigation.This bacterial strain is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) on July 12nd, 2005, and (address: No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City Institute of Microorganism, Academia Sinica), preserving number is CGMCC1412.

The construction process of genetic engineering bacterium of the present invention comprises step:

(1) starting strain is carried out mutagenic treatment with nitrosoguanidine NTG;

(2) the high transgenation bacterial strain of screening psendomonas syringae output.

The method of the invention, wherein starting strain is carried out mutagenic treatment with nitrosoguanidine NTG and comprise step:

A) cause a disease with the pseudomonas syringae soybean that to be starting strain cultivate containing on the MG substratum of kantlex in mutation (P.syringae pv.glycinea); Cultured starting strain is cultivated on shaking table; Centrifugal, the suspension thalline, concentration is 10 ⁶-10 ⁷C.f.u.ml ^-1

B) ultraviolet pre-treatment;

C) nitrosoguanidine NTG mutagenesis;

D) termination reaction, centrifugal, thalline is suspended again with the MG liquid nutrient medium.

The method of the invention, wherein screen the high transgenation bacterial strain of psendomonas syringae output and comprise step:

A) step (1) gained culture is diluted with sterilized water, be applied on the MG solid medium that contains kantlex; The single bacterium colony that grows is transferred on the new solid medium that contains kantlex cultivates;

B) primary dcreening operation: step a) gained culture is inoculated in the MG liquid nutrient medium that contains kantlex, on shaking table, cultivates; Be transferred to again in the HSC liquid nutrient medium, cultivate, extract psendomonas syringae at 16 ℃ of-20 ℃ of bottom fermentations of temperature; Screen with psendomonas syringae output;

C) inoculation that multiple sieve is high with step b) gained psendomonas syringae output is cultivated on shaking table in the MG liquid nutrient medium that contains kantlex, is transferred in the HSC liquid nutrient medium again, in 18 ℃ of fermentation culture of temperature, extracts psendomonas syringae; Screen with psendomonas syringae output.

Details are as follows for the method for the invention:

(1) starting strain is carried out mutagenic treatment with nitrosoguanidine NTG;

A) be starting strain with the mutation (P.syringae pv.glycinea) of causing a disease of pseudomonas syringae soybean, cultivate containing on the MG substratum of kantlex; Cultured starting strain is cultivated on shaking table, when the OD value reaches about 0.7; Centrifugal, abandoning supernatant is washed the gained thalline 4-5 time with the phosphoric acid buffer of pH 7.0, the suspension thalline, and making concentration is 10 ⁶-10 ⁷C.f.u.ml ^-1

B) ultraviolet pre-treatment: 15W sterilization ultraviolet lamp, 33 centimetres of distances were shone 10 seconds.

C) nitrosoguanidine NTG mutagenesis: get step a) bacterium liquid 1.8ml, add 0.2ml 0.1mg/mlNTG, reacted 10 minutes.The wherein preparation of nitrosoguanidine: behind the small amount of acetone hydrotropy, add the phosphate buffer solution (buffered soln: the NTG acetone soln is 9: 1, NTG mother liquid concentration 1mg/ml) of pH 7.0 then.

D) termination reaction: with the citrate buffer solution dilute reaction solution of 2ml pH 5.6 with termination reaction, and washing 2-3 time, centrifugal, will precipitate usefulness equal-volume MG liquid nutrient medium (KeanePJ, Kerr A, New PB.1970) and suspend again.

(2) the high transgenation bacterial strain of screening psendomonas syringae output

A) with sterilized water dilution 80-120 doubly with step (1) gained culture, be applied to (kantlex concentration is 5-15 μ g/mL) on the MG solid medium that contains kantlex, cultivated 5-10 days down for 28 ℃-30 ℃ in temperature, the single bacterium colony that grows is transferred to (kantlex concentration is 5-15 μ g/mL) on the new solid medium that contains kantlex, under 28 ℃ of-30 ℃ of conditions of temperature, cultivated 24-36 hour; The culture that obtains is a mutant strain.

Preferably, step (1) gained culture is diluted 100 times with sterilized water, be applied on MG solid medium (containing kantlex concentration the is 10 μ g/mL) flat board, in temperature is to cultivate 7-8 days under 28 ℃ of conditions, the single bacterium colony that grows is transferred on new MG solid medium (containing kantlex concentration the is 10 μ g/mL) flat board, is to cultivate 48 hours under 30 ℃ of conditions in temperature.

B) primary dcreening operation: step a) gained culture is inoculated in MG (kantlex concentration the is 5-15 μ g/mL) liquid nutrient medium, on shaking table, cultivates, be transferred to HSC liquid nutrient medium (David, 1993) in, in 18 ℃ of fermentation culture of temperature, extract psendomonas syringae, screen with psendomonas syringae output.

Preferably, step a) gained culture is inoculated in the liquid MG substratum (kantlex concentration is 10 μ g/mL),, cultivated about 24 hours on the rotating speed 260rpm/min shaking table 28 ℃ of temperature, reach at 0.6 o'clock to the OD value, be transferred in the HSC substratum by 2% inoculum size,, cultivated 7 days on the rotating speed 280rpm/min shaking table 18 ℃ of temperature, extract psendomonas syringae, measure psendomonas syringae content with high performance liquid chromatography (Palmer and Bender, 1993), screen with psendomonas syringae output.

More preferably, step a) gained culture is inoculated on the HSC liquid nutrient medium (kantlex concentration is 10 μ g/mL), culture condition: in the 20ml test tube, contain 5ml HSC substratum, tilt 45 ° to be fixed on the shaking table, temperature is 18 ℃, and rotating speed 280rpm/min cultivated 7 days, extract psendomonas syringae, screen with psendomonas syringae output.

C) multiple sieve: with the high inoculation more than 1 times of step b) gained psendomonas syringae rate ratio starting strain in MG (kantlex concentration is 10 μ g/mL) liquid nutrient medium, on shaking table, cultivate, be transferred in the HSC liquid nutrient medium, in 18 ℃ of fermentation culture of temperature, extract psendomonas syringae, screen with psendomonas syringae output.

Preferably, the high inoculation more than 1 times of step b) gained psendomonas syringae rate ratio starting strain in MG (kantlex concentration is 10 μ g/mL) liquid nutrient medium, is cultivated on shaking table, cultivated 24 hours down at 28 ℃; Be linked in the 500mL triangular flask that the 100mlHSC liquid culture is housed according to 2% inoculum size, 18 ℃ of temperature, the top fermentation of rotating speed 280rpm/min shaking table was cultivated 7 days, extracted psendomonas syringae, screened with psendomonas syringae output again.

The purposes of genetic engineering bacterium of the present invention is characterized in that described bacterial strain is used for the fermentative production of psendomonas syringae.

The present invention handles the pathogenic mutation of starting strain pseudomonas syringae soybean by the basic guanidine of inferior pin, obtain mutant, the pathogenic mutation CGMCC 1412 of the new bacterial strain pseudomonas syringae soybean that suddenlys change can improve psendomonas syringae output, and stable high yield, output can reach 100mg/L, production peak (Ullrich, 2000) than present bibliographical information is high 2.5 times.Yield poorly and unsteady problem thereby solved.

In different steps, adopt different substratum with engineering strain fermentative production psendomonas syringae provided by the invention: MG liquid nutrient medium, MG solid medium, HSC liquid nutrient medium, it is specifically composed as follows:

The MG liquid nutrient medium:

N.F,USP MANNITOL 1%

Sodium Glutamate 0.2%

KH ₂PO ₄ 0.05％

NaCl 0.02％

MgSO ₄·7H ₂O 0.02％

H ₂The O surplus

pH 7.0

The MG solid medium:

The MG liquid nutrient medium adds 1.5% agar.

The HSC substratum:

NH ₄Cl 1.0g

MgSO ₄·7H ₂O 0.2g

KH ₂PO ₄ 4.1g

K ₂HPO ₄ 3.6g

KNO ₃ 0.3g

2mM?FeCl ₃ 10ml

H ₂O 890ml

pH 6.8

Biomaterial preservation explanation

Genetic engineering bacterium pseudomonas syringae soybean of the present invention mutation (Pseudomonassyringae pv.glycinea) CGMCC 1412 that causes a disease, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC), address: Zhongguancun, Beijing City, deposit number: CGMCC1412, preservation date: on July 12nd, 2005.

Embodiment

Embodiment 1:

With the mutation (P.syringae pv.glycinea) of causing a disease of pseudomonas syringae soybean be starting strain 28 ℃ of cultivations 48 hours on the MG solid medium that contains kantlex (10 μ g/mL); Cultured starting strain is inoculated in the MG liquid nutrient medium (the kantlex final concentration is 10 μ g/mL) 28 ℃, the cultivation of 260rpm shaking table.When the OD value reached 0.7 left and right sides, centrifugal, abandoning supernatant was washed the gained thalline 4-5 time with the phosphoric acid buffer of pH value 7.0, the suspension thalline, and making cell concentration is 10 ⁷C.f.u.ml ^-1

With the 15W ultraviolet lamp of sterilizing, 33 centimetres of distances were shone 10 seconds with above-mentioned suspension bacteria liquid; Get postradiation bacterium liquid 1.8ml, add 0.2ml 0.1mg/ml NTG, mutagenesis reaction 10 minutes; The citrate buffer solution dilute reaction solution that adds 2ml pH 5.6 is with termination reaction, and washing 2-3 time, and is centrifugal, will precipitate with equal-volume MG liquid nutrient medium to suspend again; With 100 times of sterilized water dilutions, be applied on MG solid medium (containing kantlex concentration the is 10 μ g/mL) flat board, in temperature is to cultivate 7-8 days under 28 ℃ of conditions, the single bacterium colony that grows is transferred on new MG solid medium (containing kantlex concentration the is 10 μ g/mL) flat board, is to cultivate 48 hours under 30 ℃ of conditions in temperature; What obtain is mutant.

With cultured colony inoculation in MG liquid nutrient medium (kantlex concentration is 10 μ g/mL), in temperature is 28 ℃, cultivate the OD value on the rotating speed 260rpm/min shaking table and reach at 0.7 o'clock, be transferred in the HSC substratum by 2% inoculum size, 18 ℃ of temperature, cultivated 7 days on the rotating speed 280rpm/min shaking table, extract psendomonas syringae; The psendomonas syringae that extracts carries out high performance liquid chromatography and detects with chromatogram eluent dissolve with methanol; Psendomonas syringae output is that the new bacterial strain of 112mg/L is the pathogenic mutation CGMCC 1412 of pseudomonas syringae soybean.

Embodiment 2:

Pathogenic mutation CGMCC 1412 is inoculated in the MG liquid nutrient medium with new bacterial strain pseudomonas syringae soybean, in temperature is 28 ℃, cultivate the OD value on the rotating speed 260rpm/min shaking table and reach at 0.7 o'clock, be linked in the 500mL triangular flask that 100ml HSC liquid culture is housed according to 2% inoculum size again, 18 ℃ of temperature, the top fermentation of rotating speed 280rpm/min shaking table was cultivated 7 days, extracted psendomonas syringae, triplicate, psendomonas syringae output is respectively 90.1mg/L, 84.7mg/L, 92.4mg/L.

Embodiment 3:

Pseudomonas syringae soybean of the present invention is caused a disease single colony inoculation of mutation CGMCC 1412 in the test tube that MG liquid nutrient medium (kantlex concentration is 10 μ g/mL) is housed, 28 ℃ of temperature, cultivated 36 hours on the rotating speed 260rpm/min shaking table, be primary seed solution;

200ml MG liquid nutrient medium (kantlex concentration is 10 μ g/mL) is sub-packed in three 500ml triangular flasks, cultured primary seed solution is inoculated in respectively in above-mentioned three 500ml triangular flasks by 2% inoculum size, 28 ℃ of temperature, cultivated 48 hours on the rotating speed 260rpm/min shaking table, be secondary seed solution;

With the HSC substratum of dress 5L in the fermentor tank of 7L, send out sterilization with conventional hot high pressure steam sterilizing after, inoculate secondary seed solution, aeration-agitation by 2% inoculum size; 18 ℃ of leavening temperatures, fermentation pH 6.8; Fermentation time reaches 6 days at present jar, and following jar of fermented liquid be with macrolattice resin adsorption method recovery product, and uses the acetone wash-out, through recrystallization, obtains colourless crystallization shape psendomonas syringae product, and output is 89.9mg/L.

Claims (5)

1, a kind of genetic engineering bacterium, this bacterial strain is pathogenic mutation (the Pseudomonas syringae pv.glycinea) CGMCC 1412 of pseudomonas syringae soybean.

2, the construction process of the described genetic engineering bacterium of claim 1 comprises step:

(1) starting strain is carried out mutagenic treatment with nitrosoguanidine NTG;

(2) the high transgenation bacterial strain of screening psendomonas syringae output.

3,, wherein starting strain is carried out mutagenic treatment with nitrosoguanidine NTG and comprises step according to the described method of claim 2:

A) cause a disease with the pseudomonas syringae soybean that to be starting strain cultivate containing on the MG substratum of kantlex in mutation (P.syringae pv.glycinea); Cultured starting strain is cultivated on shaking table; Centrifugal, the suspension thalline, concentration is 10 ⁶-10 ⁷C.f.u.ml ^-1

B) ultraviolet pre-treatment;

C) nitrosoguanidine NTG mutagenesis;

D) termination reaction, centrifugal, thalline is suspended again with the MG liquid nutrient medium.

4,, wherein screen the high transgenation bacterial strain of psendomonas syringae output and comprise step according to the described method of claim 2:

A) step (1) gained culture is diluted with sterilized water, be applied on the MG solid medium that contains kantlex; The single bacterium colony that grows is transferred on the new solid medium that contains kantlex cultivates;

B) primary dcreening operation: step a) gained culture is inoculated in the MG liquid nutrient medium that contains kantlex, on shaking table, cultivates; Be transferred to again in the HSC liquid nutrient medium, cultivate, extract psendomonas syringae at 16 ℃ of-20 ℃ of bottom fermentations of temperature; Screen with psendomonas syringae output;

C) inoculation that multiple sieve is high with step b) gained psendomonas syringae output is cultivated on shaking table in the MG liquid nutrient medium that contains kantlex, is transferred in the HSC liquid nutrient medium again, in 18 ℃ of fermentation culture of temperature, extracts psendomonas syringae; Screen with psendomonas syringae output.

5, the purposes of the described genetic engineering bacterium of claim 1 is characterized in that described bacterial strain is used for the fermentative production of psendomonas syringae.