CN1673353A - A genetically engineered bacteria, its constructing method and use thereof - Google Patents
A genetically engineered bacteria, its constructing method and use thereof Download PDFInfo
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- CN1673353A CN1673353A CN 200510011466 CN200510011466A CN1673353A CN 1673353 A CN1673353 A CN 1673353A CN 200510011466 CN200510011466 CN 200510011466 CN 200510011466 A CN200510011466 A CN 200510011466A CN 1673353 A CN1673353 A CN 1673353A
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Abstract
The present invention relates to one kind of genetic engineering bacteria, one kind of soybean disease causing variety of clove pseudomonads with preservation number of CGMCC 1276, and its construction process and use. The genetic engineering bacteria is obtained through inserting Tn5 transposon S17-1 into the genome of the initial strain to deactivate its temperature controlling gene. The strain may be used in industrial fermentation production of crown bacterin at 26-28 deg.c, in the yield near the initial bacteria and up to 35-40 mg/L.
Description
Technical field
The invention belongs to microbial technology field.Specifically, the present invention relates to a kind of genetic engineering bacterium, its construction process and its purposes.
Background technology
Psendomonas syringae C
18H
25NO
4, molecular weight is 319, is linked with amido bond by two components of hat bacterium acid (coronafacic acid) that contain an amino acid whose hat alkanoic acid (coronamic acid) and a polyketone structure.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).Psendomonas syringae belongs to the pure natural product, on producing, uses can not pollute to environment, and be a kind of environmentally friendly biological regulator.But the research for psendomonas syringae rests on laboratory stage always at present.Reason is that its fermentative production cost is too high, does not have product in the world, and just the Individual testwas chamber is in order to study needs, purifying small amount of sample.The greatest factor that limits its industrialization lacks the strain excellent of high yield under the normal temperature (26 ℃-28 ℃) exactly.
Cause a disease the pseudomonas syringae soybean to produce the amount and the temperature of psendomonas syringae closely related in mutation (Pseudomonassyringae pv.glycinea) about studies show that, output is the highest in the time of 18 ℃, about 20mg/l, 24 ℃ of output only are 25%, almost detect during to 28 ℃ less than psendomonas syringae (David, 1993).Ullrich etc. think that the enzyme system of synthetic psendomonas syringae is subjected to temperature controlled (budde et.al 1998; Ullrich et.al 1993; Bender et.al 1996:Rangaswamyet.al 1997; Ullrich, 2000).
At present, produce psendomonas syringae can only so then will consume resources such as a large amount of electricity, water and be used for temperature control with 18 ℃ low temperature fermentation.The short national conditions of this and china natural resources are not corresponding.Also greatly improved production cost.
Summary of the invention
The objective of the invention is to obtain a kind of engineering strain of fermentative production psendomonas syringae at normal temperatures that can be used in.
Another object of the present invention provides the construction process of this bacterial strain.
A further object of the present invention provides the purposes 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 temperature controlling gene sudden change inactivation, 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 December 21st, 2004, and (address: No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City Institute of Microorganism, Academia Sinica), preserving number is CGMCC1276.
The method that makes up the pathogenic mutation CGMCC1276 of pseudomonas syringae soybean of the present invention comprises the steps:
1, Tn5 transposon S17-1 is inserted in the genome of the bacterium that sets out;
2, the bacterial strain of screening temperature controlling gene sudden change inactivation obtains described bacterial strain, obtains described bacterial strain.
Details are as follows for described method:
1, Tn5 transposon S17-1 is inserted in the genome of the bacterium that sets out;
Choose plasmid vector pRL1063a, wherein have Tn5 transposon S17-1 (as Fig. 1).Plasmid vector pRL1063a and starting strain (pseudomonas syringae soybean cause a disease mutation (P.syringae pv.glycinea)) are cultivated containing on the substratum of kantlex respectively; Good plasmid vector pRL1063a and the starting strain cultivated are mixed, on shaking table, cultivate, Tn5 transposon S17-1 is inserted in the genome of starting strain; Centrifugal, abandoning supernatant is cultivated the gained bacterial sediment.
The culture that obtains is the mixture of the mutant strain of plasmid vector pRL1063a, starting strain, insertion transposon S17-1.
This step is preferably cultivated plasmid vector pRL1063a and starting strain containing on the LB substratum that concentration is 5 μ g/L-15 μ g/L kantlex respectively; By volume 1: 1-2 gets plasmid vector pRL1063a and starting strain mixes, in temperature is 26 ℃-32 ℃, rotating speed is to act on 20-40 minute on the 150rpm/min-300rpm/min shaking table, with the centrifugal 5-15 of 10000r/min-20000r/min minute, abandoning supernatant, bacterial sediment is inoculated on the LB solid medium, is to cultivate 12-48 hour under 26 ℃ of-32 ℃ of conditions in temperature.
This step is more preferably cultivated plasmid vector pRL1063a and starting strain containing on the LB substratum that concentration is 10 μ g/L kantlex respectively; Plasmid vector pRL1063a and the starting strain of getting the growth logarithmic phase in 1: 1.2 by volume mix, in temperature is 30 ℃, rotating speed is to act on 30 minutes on the 200rpm/min shaking table, with the centrifugal 10min of 12000r/min, use the physiological saline washed twice, supernatant discarded night is inoculated into bacterial sediment on the LB solid medium, is to cultivate 24 hours under 30 ℃ of conditions in temperature.
2, the bacterial strain of screening temperature controlling gene sudden change inactivation, this step comprises following three phases:
(1) step 1 gained culture is suspended with liquid nutrient medium, be applied on the solid medium that contains kantlex and Streptomycin sulphate simultaneously and cultivate; The single bacterium colony that grows is transferred on the solid medium that contains kantlex and Streptomycin sulphate new the time cultivates.
The culture that obtains is the mutant strain of plasmid vector pRL1063a and insertion transposon S17-1.
This step preferably suspends step 1 gained culture with the MG liquid nutrient medium, be applied on the MG solid medium (kantlex concentration is that 5 μ g/L-15 μ g/L, Streptomycin sulphate concentration are 20 μ g/L-40 μ g/L), in temperature is to cultivate 5-10 days under 26 ℃ of-32 ℃ of conditions, the single bacterium colony that grows is transferred on the new MG solid medium (kantlex concentration is that 5 μ g/L-15 μ g/L, Streptomycin sulphate concentration are 20 μ g/L-40 μ g/L), is to cultivate 24-36 hour under 26 ℃ of-32 ℃ of conditions in temperature.
This step more preferably suspends step 1 gained culture with the MG liquid nutrient medium, be applied to MG solid medium (Keane PJ, Kerr A, New PB.1970) on (kantlex concentration is that 10 μ g/L, Streptomycin sulphate concentration are 30 μ g/L) flat board, in temperature is to cultivate 7-8 days under 30 ℃ of conditions, the single bacterium colony that grows is transferred on new MG solid medium (kantlex concentration is that 10 μ g/L, Streptomycin sulphate concentration the are 30 μ g/L) flat board, is to cultivate 48 hours under 30 ℃ of conditions in temperature.
(2) be that goal gene carries out pcr amplification with step (1) gained culture with the luxAB gene.Amplification gene is luxAB, primer P1:5 '-TTT GTT CGG CTT GGT ATCGC-3 '; P2:5 '-ATT GCT TAG GTC CAT TCT CA-3 '.Choosing the bacterium colony (as Fig. 2) that can expand target gene fragment cultivates;
The gained culture is for inserting the mutant strain of transposon S17-1.
This step is that goal gene carries out pcr amplification with step (1) gained culture with the luxAB gene preferably, chooses the bacterium colony that can expand target gene fragment, is to cultivate 12-48 hour under 26 ℃ of-32 ℃ of conditions in temperature;
This step is that goal gene carries out pcr amplification with step (1) gained culture with the luxAB gene more preferably, chooses the bacterium colony that can expand target gene fragment, be seeded on the MG solid medium, and be to cultivate (12-48 hour) under 30 ℃ of conditions in temperature.
(3) step (2) gained culture is inoculated in the liquid nutrient medium that contains kantlex and Streptomycin sulphate simultaneously, on shaking table, cultivates, be transferred in the new substratum,, choose the bacterial strain that to produce psendomonas syringae in 26 ℃ of-28 ℃ of fermentation culture of temperature.
This step preferably is inoculated into step (2) gained culture in the MG liquid nutrient medium (kantlex concentration is that 5 μ g/L-15 μ g/L, Streptomycin sulphate concentration are 20 μ g/L-40 μ g/L), 26 ℃-30 ℃ of temperature, cultivate the OD value on the rotating speed 150rpm/min-300rpm/min shaking table when reaching 0.6-0.8,1%-3% by inoculum size is transferred to HSC (David, 1993) in the substratum, 26 ℃-28 ℃ of temperature, cultivated 2-10 days on the rotating speed 150rpm/min-300rpm/min shaking table, choose the bacterial strain that to produce psendomonas syringae.
This step more preferably is inoculated into step (2) gained culture in the MG liquid nutrient medium (kantlex concentration is that 10 μ g/L, Streptomycin sulphate concentration are 30 μ g/L), 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% of inoculum size, 28 ℃ of temperature, cultivated 5 days on the rotating speed 260rpm/min shaking table, choose the bacterial strain that to produce psendomonas syringae.
The plasmid vector pRL1063a that the present invention selects for use, its advantage is to contain luxAB gene (bacterium does not have), and contains a plurality of selective markers and insert site etc.
The Tn5 transposon S17-1 that the present invention selects for use contains kalamycin resistance gene and streptomycin resistance gene, and this transposon is safe to environment.
The genetic engineering bacterium of temperature control transgenation inactivation provided by the invention can be used for normal temperature fermentation and produces psendomonas syringae.
Different steps producing psendomonas syringae with strain fermentation provided by the invention, can adopt following different substratum: A substratum, B solid medium, B liquid nutrient medium, C substratum, it is specifically composed as follows:
The A substratum:
Peptone 1%
Yeast extract 0.5%
NaCl 0.5％
Agar 1.5%
pH 7.0
The B 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％
pH 7.0
The B solid medium:
The B liquid nutrient medium adds 1.5% agar.
The C substratum:
NH
4C1 1.0g
MgSO
4·7H
2O 0.2g
KH
2PO
4 4.1g
K
2HPO
4 3.6g
KNO
3 0.3g
2mMFeCl
3 10ml
H
2O 890ml
pH 6.8
The present invention is inserted into transposon S17-1 on the temperature adjusting gene of starting strain by Tn5 transposon mutant method, make this transgenation inactivation, mutation CGMCC1276 can produce psendomonas syringae down at normal temperature (26 ℃-28 ℃) thereby the new bacterial strain pseudomonas syringae soybean that makes sudden change is caused a disease, and stable high yield, output can reach 35mg/L-40mg/L, thereby has solved the expensive problem of 18 ℃ of low temperature fermentations.
Biomaterial preservation explanation
Pseudomonas syringae soybean mutation (Pseudomonas syringaepv.glycinea) CGMCC1276 that causes a disease, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC), address: No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City Institute of Microorganism, Academia Sinica, deposit number: CGMCC1276, preservation date: on December 21st, 2004.
Description of drawings
Fig. 1 is a plasmid PRL1063a structural representation.
Fig. 2 is PCR figure as a result.Swimming lane M is a dna molecular amount mark, and swimming lane 1 is a plasmid vector for pRL1063a, and swimming lane 2-10 is for inserting the mutant strain of transposon S17-1.
Embodiment
Embodiment 1
Plasmid vector pRL1063a and starting strain are cultivated containing on the LB substratum that concentration is 10 μ g/L kantlex respectively; Plasmid vector pRL1063a and the starting strain of getting the growth logarithmic phase in 1: 1.2 by volume mix, in temperature is 30 ℃, rotating speed is to act on 30 minutes on the 200rpm/min shaking table, the centrifugal 10min of 12000r/min, use the physiological saline washed twice, supernatant discarded night is inoculated into bacterial sediment on the LB solid medium, is to cultivate 24 hours under 30 ℃ of conditions in temperature.The culture that obtains is the mixture of the mutant strain of plasmid vector pRL1063a, starting strain, insertion transposon S17-1.
Cultured bacterial sediment is scraped with toothpick, suspend with the MG liquid nutrient medium, be applied on MG solid medium (kantlex concentration is that 10 μ g/L, Streptomycin sulphate concentration the are 30 μ g/L) flat board, in temperature is to cultivate 7-8 days under 30 ℃ of conditions, the single bacterium colony that grows is transferred on new MG solid medium (kantlex concentration is that 10 μ g/L, Streptomycin sulphate concentration the are 30 μ g/L) flat board, is to cultivate 48 hours under 30 ℃ of conditions in temperature.The culture that obtains is the mixture of the mutant strain of plasmid vector pRL1063a, insertion transposon S17-1.
With the luxAB gene is that goal gene carries out pcr amplification, primer P1:5 '-TTT GTTCGG CTT GGT ATC GC-3 '; P2:5 '-ATT GCT TAG GTC CAT TCTCA-3 ' chooses the bacterium colony (as Fig. 2) that can expand target gene fragment, is to cultivate 24 hours under 30 ℃ of conditions in temperature.The gained culture is for inserting the mutant strain of transposon S17-1.
The good bacterium colony of inoculation culture is in MG liquid nutrient medium (kantlex concentration is that 10 μ g/L, Streptomycin sulphate concentration are 30 μ g/L), in temperature is 28 ℃, cultivate the OD value on the rotating speed 260rpm shaking table and reach at 0.7 o'clock, be transferred in the HSC substratum by 2% of inoculum size, 28 ℃ of temperature, cultivated 5 days on the rotating speed 260rpm/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 37.5mg/l.
Embodiment 2
Plasmid vector pRL1063a and starting strain are cultivated containing on the LB substratum that concentration is 5 μ g/L kantlex respectively; Plasmid vector pRL1063a and the starting strain of getting the growth logarithmic phase in 1: 1 by volume mix, in temperature is 26 ℃, rotating speed is to act on 20 minutes on the 150rpm/min shaking table, with the centrifugal 5min of 20000r/min, with the physiological saline washing once, supernatant discarded night is inoculated into bacterial sediment on the LB solid medium, is to cultivate 12 hours under 26 ℃ of conditions in temperature.The culture that obtains is the mixture of the mutant strain of plasmid vector pRL1063a, starting strain, insertion transposon S17-1.
Cultured bacterial sediment is scraped with toothpick, suspend with the MG liquid nutrient medium, be applied on MG solid medium (kantlex concentration is that 5 μ g/L, Streptomycin sulphate concentration the are 20 μ g/L) flat board, in temperature is to cultivate 5 days under 26 ℃ of conditions, the single bacterium colony that grows is transferred on new MG solid medium (kantlex concentration is that 5 μ g/L, Streptomycin sulphate concentration the are 20 μ g/L) flat board, is to cultivate 24 hours under 26 ℃ of conditions in temperature.The culture that obtains is the mixture of the mutant strain of plasmid vector pRL1063a, insertion transposon S17-1.
With the luxAB gene is that goal gene carries out pcr amplification, chooses the bacterium colony (as Fig. 2) that can expand target gene fragment, is to cultivate 12 hours under 26 ℃ of conditions in temperature; The gained culture is for inserting the mutant strain of transposon S17-1.
The good bacterium colony of inoculation culture is in MG liquid nutrient medium (kantlex concentration is that 5 μ g/L, Streptomycin sulphate concentration are 20 μ g/L), in temperature is 26 ℃, cultivate the OD value on the rotating speed 150rpm/min shaking table and reach at 0.6 o'clock, be transferred in the HSC substratum by 1% of inoculum size, 26 ℃ of temperature, cultivated 2 days on the rotating speed 150rpm/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 35mg/l.
Embodiment 3
Plasmid vector pRL1063a and starting strain are cultivated containing on the LB substratum that concentration is 15 μ g/L kantlex respectively; Plasmid vector pRL1063a and the starting strain of getting the growth logarithmic phase in 1: 2 by volume mix, in temperature is 32 ℃, rotating speed is to act on 40 minutes on the 300rpm/min shaking table, the centrifugal 15min of 10000r/min, supernatant discarded night, bacterial sediment is inoculated on the LB solid medium, is to cultivate 48 hours under 32 ℃ of conditions in temperature.The culture that obtains is the mixture of the mutant strain of plasmid vector pRL1063a, starting strain, insertion transposon S17-1.
Cultured bacterial sediment is scraped with toothpick, MG suspends with liquid nutrient medium, be applied on MG solid medium (kantlex concentration is that 15 μ g/L, Streptomycin sulphate concentration the are 40 μ g/L) flat board, in temperature is to cultivate 10 days under 32 ℃ of conditions, the single bacterium colony that grows is transferred on new MG solid medium (kantlex concentration is that 15 μ g/L, Streptomycin sulphate concentration the are 40 μ g/L) flat board, is to cultivate 36 hours under 32 ℃ of conditions in temperature.The culture that obtains is the mixture of the mutant strain of plasmid vector pRL1063a, insertion transposon S17-1.
With the luxAB gene is that goal gene carries out pcr amplification, chooses the bacterium colony (as Fig. 2) that can expand target gene fragment, is to cultivate 48 hours under 32 ℃ of conditions in temperature; The gained culture is for inserting the mutant strain of transposon S17-1.
The good bacterium colony of inoculation culture is in MG liquid nutrient medium (kantlex concentration is that 15 μ g/L, Streptomycin sulphate concentration are 40 μ g/L), in temperature is 30 ℃, cultivate the OD value on the rotating speed 300rpm/min shaking table and reach at 0.8 o'clock, be transferred in the HSC substratum by 3% of inoculum size, 28 ℃ of temperature, cultivated 10 days on the rotating speed 300rpm/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 40mg/l.
Embodiment 4
Single bacterium colony of the pathogenic mutation CGMCC1276 of inoculation pseudomonas syringae soybean 26 ℃ of temperature, is cultivated seed liquor on the rotating speed 260rpm/min shaking table in the test tube that B liquid nutrient medium (kantlex concentration is that 10 μ g/L, Streptomycin sulphate concentration are 30 μ g/L) is housed.When seed liquor OD value reaches 0.7, getting 1ml is transferred in 500ml triangular flask, the interior dress 50mlC substratum (kantlex concentration is that 10 μ g/L, Streptomycin sulphate concentration are 30 μ g/L), 26 ℃ of temperature, cultivated 5 days on the rotating speed 260rpm/min shaking table, extract psendomonas syringae.Carry out three re-treatments.Carry out high performance liquid chromatography and detect, be confirmed to be psendomonas syringae, output reaches 37.5mg/l.
Embodiment 5
Single bacterium colony of the pathogenic mutation CGMCC1276 of inoculation pseudomonas syringae soybean is in the test tube that B liquid nutrient medium (kantlex concentration is that 10 μ g/L, Streptomycin sulphate concentration are 30 μ g/L) is housed, 28 ℃ of temperature, cultivated 36-48 hour on the rotating speed 260rpm/min shaking table, be primary seed solution.
250ml B liquid nutrient medium is sub-packed in three 500ml triangular flasks.Cultured primary seed solution is inoculated in respectively in above-mentioned three 500ml triangular flasks by the 1-2% inoculum size,, cultivated 36-48 hour on the rotating speed 260rpm/min shaking table, be secondary seed solution 28 ℃ of temperature.
With the C substratum of adorning 5 liters in 7 liters the fermentor tank, send out sterilization with conventional hot high pressure steam sterilizing after, press 1-2% inoculum size inoculation secondary seed solution, aeration-agitation.28 ℃ of leavening temperatures, fermentation pH5.5-6.8.
Fermentation time reaches 5 days at present jar.Following jar of fermented liquid reclaims product with the macrolattice resin adsorption method, and uses ethanol elution, through recrystallization, obtains colourless crystallization shape psendomonas syringae product, and output is 37.3mg/l.
Claims (4)
1, a kind of genetic engineering bacterium, this bacterial strain is pathogenic mutation (the Pseudomonas syringae pv.glycinea) CGMCC 1276 of pseudomonas syringae soybean.
2, the construction process of the described genetic engineering bacterium of claim 1, this method comprises the steps:
(1) Tn5 transposon S17-1 is inserted in the genome of the bacterium that sets out;
(2) bacterial strain of screening temperature controlling gene sudden change inactivation.
3, the method for claim 2, the bacterial strain step of wherein said screening temperature controlling gene sudden change inactivation comprises following three phases:
A) step (1) gained culture is suspended with liquid nutrient medium, be applied on the solid medium that contains kantlex and Streptomycin sulphate simultaneously and cultivate; The single bacterium colony that grows is transferred on the solid medium that contains kantlex and Streptomycin sulphate new the time cultivates;
B) be that goal gene carries out pcr amplification with step a) gained culture with the luxAB gene, choose the bacterium colony that can expand target gene fragment and cultivate;
C) step b) gained culture is inoculated in the liquid nutrient medium that contains kantlex and Streptomycin sulphate simultaneously, on shaking table, cultivates, be transferred in the new substratum,, choose the bacterial strain that to produce psendomonas syringae in 26 ℃ of-28 ℃ of fermentation culture of temperature.
4, 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.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101033457B (en) * | 2007-02-16 | 2010-12-15 | 江西农业大学 | Method of producing coronatine by burkholderiacepacia and fermentation culture medium thereof |
| CN102293201A (en) * | 2011-06-20 | 2011-12-28 | 中国农业大学 | New purpose of coronatine |
-
2005
- 2005-03-23 CN CN 200510011466 patent/CN1281738C/en active Active
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101033457B (en) * | 2007-02-16 | 2010-12-15 | 江西农业大学 | Method of producing coronatine by burkholderiacepacia and fermentation culture medium thereof |
| CN102293201A (en) * | 2011-06-20 | 2011-12-28 | 中国农业大学 | New purpose of coronatine |
| CN102293201B (en) * | 2011-06-20 | 2015-07-01 | 中国农业大学 | New purpose of coronatine |
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