CN1539959A - Method for inserting gene of pseudomonas fluorescens M18 into mutation strain H18G - Google Patents
Method for inserting gene of pseudomonas fluorescens M18 into mutation strain H18G Download PDFInfo
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- CN1539959A CN1539959A CNA2003101083022A CN200310108302A CN1539959A CN 1539959 A CN1539959 A CN 1539959A CN A2003101083022 A CNA2003101083022 A CN A2003101083022A CN 200310108302 A CN200310108302 A CN 200310108302A CN 1539959 A CN1539959 A CN 1539959A
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Abstract
A method for inserting the gene of fluorescent pseudomonads M18 in the mutant strain M18G in order to develop efficient low-poison agricultural chemical includes designing facultative primer, PCR amplification of gacA fragment in M18 genon, using it as probe to screen positive clone from the plasmid library of M18 genom, using homogeneous recombination reject technique and solid-phase filter membrane triparent cross to obtain mutant strain M18G, and extracting and quantitatively testing phenazine-1-carboxylate.
Description
Technical field
What the present invention relates to is the method that a kind of pseudomonas gene inserts mutant strain, and the gene of especially a kind of Pseudomonas fluorescens M18 inserts the method for mutant strain M18G, belongs to the gene engineering field.
Background technology
It is generally acknowledged that the underproduction amplitude that corps diseases causes accounts for the 25-75% of gross income.At present, except the cultivation step of selecting breeding and science for use, the main means of control corps diseases are to use chemical bactericide in the production.Mostly sterilant is harmful to animal and human's class, and the food poisoning that chemical pesticide caused happens occasionally, and residual poisonous substance has caused the concern of social each side to people and animals' potential hazard in the food of edible portion.The chemical pesticide of long-term accumulation in the ecosystem caused the pollution of environment, and be totally unfavorable to the Sustainable development of national economy.Simultaneously, in many cases, chemical pesticide is not in full force and effect.Therefore, in the chemical pesticide of new generation of the compatible high-efficiency low-toxicity of development environment, further develop microbial pesticide and seem very necessary.
According to bibliographical information (microorganism journal, 2003,43 (3): 315-323), utilize the amplification technique of rpoD gene, can only make the output of phenazine-1-carboxylic acid of the M18 bacterial strain of wild-type improve about 2 times and reach 102ppm by 52ppm.Amplification is not high.
Summary of the invention
The objective of the invention is to overcome on the basis of the defective in the above-mentioned technology, the method of the gene insertion mutant strain M18G of a kind of Pseudomonas fluorescens M18 is provided, pass through genetic engineering technique, carry out orthomutation, obtain a kind of engineering strain M18G, fermentation titer can be brought up to 317ppm, improve the ability of Pseudomonas fluorescens (fluorescent Pseudomonas sp.) M18 biosynthesizing phenazine-1-carboxylic acid.
The present invention realizes by the following technical solutions:
1, polymerase chain reaction: according to intestinal bacteria E.coli, gacA gene conservative sequence in the bacterial classifications such as Pseudomonas aeruginosa P.aeruginosa and Erwinia E.carotovora has designed that to be used for Pseudomonas fluorescens M18 genome be the oligodeoxynucleotide degenerate primer that template is carried out PCR amplification.Primer sequence is: primer 1:5 '-GCCT
TCTAGATGATWARGGTGYTRGTCGAYGACCA-3 ', primer 2: 5 '-GATTAA
CTCGAGYYAGVBGSYGGCATCRACCATG-3 ' wherein annexs base and is: Y=C/T, W=A/T, V=A/C/G, R=A/G, B=G/C/T, S=G/C.5 ' end at two primers adds XhoI and XbaI enzyme cutting site (underscore position) respectively, is beneficial to the polymerase chain reaction product clone.The condition of PCR reaction is: 95 ℃ of (1) pre-sex change, 6min; (2) each circulation of amplified reaction comprises 94 ℃ of sex change, 1min, and anneal 50 ℃, 2min extend 72 ℃, 3min, amounts to 30 circulations; (3) 72 ℃ of postreactions, 10min.Obtained the part segment of gacA gene.
2, the part segment with this gacA gene is a probe, filters out positive colony from the genome Ke Si library of M18.From the endonuclease bamhi of the coemid of positive colony, obtain to contain by southern blotting gacA gene and both sides sequence 1.9kb fragment and clone in pBluescriptSK
+In.
3, will the encode sequence length of resistant gene of kantlex is the 1.6kb fragment, inserts the BamHI site that is arranged in the gacA sequence, at carrier pBluescriptSK
+On finish this gene fixed point insert sudden change.Afterwards, be length the gacA fragment of the resistant gene that is inserted with kantlex of 3.5kb, from pBluescriptSK
+Downcut and clone in suicide plasmid pME3087.
4, utilization homologous recombination technique, the mutant strain M18G of structure M18.Adopt solid phase filter membrane tri-parent conjugation method, the donor bacterium is the intestinal bacteria ED8767 of band pME3087G, and volume is 100ul, and being subjected to thalline is wild-type M18, volume 100 μ l and carry the auxiliary bacterium HB101 of helper plasmid pME497, volume 100ul mixes, 4 ℃, the centrifugal 2min of 12000r/min, remove supernatant, fresh culture drip washing secondary, resuspending is in the 100ul fresh culture.Get the 5ul point sample on aseptic filter membrane, cultivate to scrape after 18 hours and get thalline, place the 1ml nutrient solution, the suspension mixing, the resistant panel that contains 50 μ g/ml kantlex, 20 μ g/ml rifampins is coated in dilution, and whole altogether and zygote appear in 37 ℃ of cultivations behind the 12-24h.The several zygote concussions of random choose are cultivated the back dilution and are coated with flat board, put plate screening then, contrast dull and stereotyped resistance and be: kantlex, rifampin, screen dull and stereotyped resistance and be: kantlex, rifampin and tsiklomitsin, through the positive colony of secondary exchange, thereby obtain mutant strain M18G.
5, the extraction of phenazine-1-carboxylic acid and quantitative assay.Get zymocyte liquid 180 μ l, add 20 μ l 6N hcl acidifyings it, the concussion lysing cell.Add the chloroform of 3 times of volumes, shake balance 5min again.The centrifugal 5min of 12000r/min.Discard top bacterial debris.Take off 5 μ sample introductions of face, carry out the output that HPLC measures phenazine-1-carboxylic acid.The condition that detects is: detect wavelength 258nm, retention time is 1.7min; Moving phase is 70% methyl alcohol, flow velocity 1ml/min, C
18Reversed-phase column.
The present invention has significant characteristics and substantive progressive, and sheath blight fungus is a kind of common plant pathogenic fungi, causes the crop production reduction that infects.Phenazine-1-carboxylic acid can efficiently suppress the growth of sheath blight fungus, and cover crop avoids infecting.No matter mutant strain M18G is at King ' B or is improving in the PPM substratum that the synthesis capability of its phenazine-1-carboxylic acid all increases significantly.By bio-fermentation engineering, it is efficient that this bacterial strain can be used for exploitation, the novel biopesticide of low toxicity.Has comparatively wide application prospect.
Description of drawings
Fig. 1, mutant strain M18G and wild strain M18 are improving synthetic phenazine-1-carboxylic acid graphic representation under the PPM substratum.●, ◆ represent mutant strain M18G and wild strain M18 respectively.
Fig. 2, mutant strain M18G and wild strain M18 be synthetic phenazine-1-carboxylic acid graphic representation under King ' B substratum.●, ◆ represent mutant strain M18G and wild strain M18 respectively.
Embodiment
Further describe embodiment below in conjunction with technical scheme of the present invention
1, improving under the PPM substratum fermentation condition, M18 compares with wild strain, and the ability of the synthetic phenazine-1-carboxylic acid of mutant strain M18G improves 2 times.Mutant strain M18G and wild strain M18 are inoculated in respectively in the Erlenmeyer flask that contains 150ml PPM substratum, the volume of Erlenmeyer flask is 500ml, places the shaking table fermentation culture under 28 ℃, 220r/min condition, every 8 hours sampling analysis, repeat, get its mean value for 3 times.After fermentation in 72 hours, the HPLC measurement result shows: the amount of M18G biosynthesizing PCA can reach 121.5ppm, and the amount of the biosynthetic PCA of M18 is 60.6ppm only, sees Fig. 1.
2, under King ' B substratum fermentation condition, M18 compares with wild strain, and the ability of the synthetic PCA of mutant strain M18G improves nearly 30 times.Mutant strain M18G and wild strain M18 are inoculated respectively and fill in the Erlenmeyer flask of 150ml PPM substratum, and the volume of Erlenmeyer flask is 500ml, places 28 ℃, 220r/min, fermentation culture in the shaking table, every 8 hours sampling analysis, its mean value was got in 3 repetitions.After fermentation in 72 hours, the HPLC measurement result shows: the amount of M18G biosynthesizing PCA can reach 317ppm, and the output of the biosynthetic phenazine-1-carboxylic acid of wild-type M18 is 13.2ppm only, sees Fig. 2.
Claims (7)
1, the gene of a kind of Pseudomonas fluorescens M18 inserts the method for mutant strain M18G, it is characterized in that the step of concrete grammar is as follows:
(1) primer and polymerase chain reaction: according to intestinal bacteria E.coli, gacA gene conservative sequence in Pseudomonas aeruginosa P.aeruginosa and the Erwinia E.carotovora bacterial classification, the design degenerate primer is used for the genomic gacA fragment of amplification fluorescent pseudomonas M 18;
(2) be probe with this fragment, from the genome coemid library of M18, sift out positive colony;
(3) the utilization homologous recombination technique is rejected technology and solid phase filter membrane tri-parent conjugation method, obtains the gacA mutant strain M18G of M18 bacterial strain;
(4) extraction of phenazine-1-carboxylic acid and quantitative assay: get zymocyte liquid 180 μ l, add 20 μ l 6N hcl acidifyings, the concussion lysing cell, through the chloroform of 3 times of volumes, behind the concussion balance 5min, the centrifugal 5min of 12000r/min, supernatant discarded night and bacterial debris, get 5 μ l lower floor liquid phases, sample introduction is through the output of HPLC mensuration phenazine-1-carboxylic acid.
2, the gene of Pseudomonas fluorescens M18 according to claim 1 inserts the method for mutant strain M18G, it is characterized in that the described primer of step (1) specifically refers to: primer 1:5 '-GCCT
TCTAGATGATWARGGTGYTRGTCGAYGACCA-3 ', primer 2: 5 '-GATTAA
CTCGAGYYAGVBGSYGGCATCRACCATG-3 ' wherein annexs base and is: Y=C/T, W=A/T, V=A/C/G, R=A/G, B=G/C/T, S=G/C.
3, the gene of Pseudomonas fluorescens M18 according to claim 1 inserts the method for mutant strain M18G, it is characterized in that, described primer adds XhoI and XbaI enzyme cutting site at 5 ' of two primers respectively, is beneficial to clone, order-checking and the evaluation of polymerase chain product.
4, the gene of Pseudomonas fluorescens M18 according to claim 1 inserts the method for mutant strain M18G, it is characterized in that described PCR reaction conditions is:
(1) 95 ℃ of pre-sex change, 6min;
(2) each circulation of amplified reaction comprises 94 ℃ of sex change, 1min, and anneal 50 ℃, 2min extend 72 ℃, 3min, amounts to 30 circulations;
(3) 72 ℃ of postreactions, 10min, in view of the above, the partial sequence of amplification and acquisition gacA gene from the genome of M18; The fragment cloning of amplification is in pBluescriptSK
+In, corresponding plasmid is pBSG.
5, the gene of Pseudomonas fluorescens M18 according to claim 1 inserts the method for mutant strain M18G, it is characterized in that, from the coemid positive colony, cut have gacA gene and both sides sequence the 1.9kb fragment cloning in pBluescriptSK
+In, the gene of coding kalamycin resistance inserted be arranged in the nearly intermediary of this fragment and, obtain gacA gene in vitro mutant in the BamHI site of gacA gene.
6, the gene of Pseudomonas fluorescens M18 according to claim 1 inserts the method for mutant strain M18G, and it is characterized in that being further defined to of step (2): the fragment that will be inserted with kalamycin resistance gene is from pBluescriptSK
+In cut out and clone in suicide plasmid pME3087 and form pME3087G.
7, the gene of Pseudomonas fluorescens M18 according to claim 1 inserts the method for mutant strain M18G, it is characterized in that, to the extraction of step (4) and further being defined as of mensuration: the HPLC testing conditions is: it is 258nm that PCA detects wavelength, and retention time is 1.7min; Moving phase is 70% methyl alcohol, and flow velocity is 2 μ l/min, C
18Reversed-phase column.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100411521C (en) * | 2006-01-19 | 2008-08-20 | 上海交通大学 | Method for preparing bactericidal agent using antagonizing bacteria M18 strain |
| CN101705265B (en) * | 2009-11-12 | 2011-07-27 | 上海交通大学 | Method for producing phenazine-1-carboxylic acid by using engineering bacterial strain M18G to carry plasmid pME6032Phz |
| CN103834585A (en) * | 2013-10-24 | 2014-06-04 | 上海交通大学 | Rhizospheric pseudomonad capable of largely producing phenazine-1-carboxylic acid and phenazine-1-amide |
| CN104293692A (en) * | 2014-07-14 | 2015-01-21 | 武汉汉申生物科技有限责任公司 | Bioengineering bacterial strain, preparation method and application thereof |
| CN104671430A (en) * | 2014-12-16 | 2015-06-03 | 华南农业大学 | Method for efficiently degrading beta-cypermethrin by utilizing pseudomonas aeruginosa |
| CN105802872A (en) * | 2015-05-29 | 2016-07-27 | 广东双骏生物科技有限公司 | Fluorescent pseudomonas, method for producing phenazine amide and application of phenazine amide |
| CN111690700A (en) * | 2020-07-01 | 2020-09-22 | 上海农乐生物制品股份有限公司 | Shenqinmycin fermentation process |
| CN114592080A (en) * | 2022-04-01 | 2022-06-07 | 领航基因科技（杭州）有限公司 | RT-ddPCR reagent for detecting four pathogenic bacteria |
-
2003
- 2003-10-30 CN CNA2003101083022A patent/CN1539959A/en active Pending
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100411521C (en) * | 2006-01-19 | 2008-08-20 | 上海交通大学 | Method for preparing bactericidal agent using antagonizing bacteria M18 strain |
| CN101705265B (en) * | 2009-11-12 | 2011-07-27 | 上海交通大学 | Method for producing phenazine-1-carboxylic acid by using engineering bacterial strain M18G to carry plasmid pME6032Phz |
| CN103834585A (en) * | 2013-10-24 | 2014-06-04 | 上海交通大学 | Rhizospheric pseudomonad capable of largely producing phenazine-1-carboxylic acid and phenazine-1-amide |
| CN103834585B (en) * | 2013-10-24 | 2016-02-10 | 上海交通大学 | The rhizosphere pseudomonas of high yield phenazine-1-carboxylic acid and azophenlyene-1-acid amides |
| CN104293692A (en) * | 2014-07-14 | 2015-01-21 | 武汉汉申生物科技有限责任公司 | Bioengineering bacterial strain, preparation method and application thereof |
| CN104671430A (en) * | 2014-12-16 | 2015-06-03 | 华南农业大学 | Method for efficiently degrading beta-cypermethrin by utilizing pseudomonas aeruginosa |
| CN105802872A (en) * | 2015-05-29 | 2016-07-27 | 广东双骏生物科技有限公司 | Fluorescent pseudomonas, method for producing phenazine amide and application of phenazine amide |
| CN105802872B (en) * | 2015-05-29 | 2020-07-24 | 广东双骏生物科技有限公司 | Pseudomonas fluorescens, method for producing phenazine amide and application thereof |
| CN111690700A (en) * | 2020-07-01 | 2020-09-22 | 上海农乐生物制品股份有限公司 | Shenqinmycin fermentation process |
| CN111690700B (en) * | 2020-07-01 | 2022-03-18 | 上海农乐生物制品股份有限公司 | Shenqinmycin fermentation process |
| CN114592080A (en) * | 2022-04-01 | 2022-06-07 | 领航基因科技（杭州）有限公司 | RT-ddPCR reagent for detecting four pathogenic bacteria |
| CN114592080B (en) * | 2022-04-01 | 2023-12-01 | 领航基因科技（杭州）有限公司 | RT-ddPCR reagent for detecting four pathogenic bacteria |
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