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Method for producing phenazine-1-carboxylic acid by using engineering bacterial strain M18G to carry plasmid pME6032Phz

A technology of engineering strains and plasmids, applied in the field of high-efficiency production of phenazine-1-carboxylic acid and microbial source fungicides, can solve the problems of low added value and high cost, and achieve enhanced expression, increased expression, efficient and stable synthesis Effect

Active Publication Date: 2011-07-27
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, rice, the most important food crop for people in my country and Asia, is a commodity with low added value. If Shenzimycin is produced according to the current fermentation level, the cost for preventing and treating rice crop diseases is still high, and it is still difficult to produce. Accepted by food and agriculture and carried out large-scale promotion and application

Method used

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  • Method for producing phenazine-1-carboxylic acid by using engineering bacterial strain M18G to carry plasmid pME6032Phz
  • Method for producing phenazine-1-carboxylic acid by using engineering bacterial strain M18G to carry plasmid pME6032Phz

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Experimental program
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Effect test

Embodiment 1

[0032] 1. Amplify the coding gene fragment for phenazine-1-carboxylic acid biosynthesis (phzA1-G1)

[0033] Design a pair of primers to amplify the coding gene fragment (phzA1-G1) of phenazine-1-carboxylic acid biosynthesis. The nucleotide sequence of the primers is as follows:

[0034] Forward: 5’-ATATAT GGTACC GCCAGCGAATAACCGATGCCGCGAGGGAA-3’

[0035] Reverse: 5’-TGCGTA AGATCT CGATGGGTTCGCTCATGGGTGCTTCCTTTT-3’

[0036] The underline in the sequence is the restriction enzyme KpnI and BglII restriction sites. The primer was synthesized by Shanghai Shenggong Biological Engineering Technology Service Co., Ltd.

[0037] Then, using the growth-promoting antagonistic antibacterial M18 genomic DNA as a template, using DNA polymerase LA Taq and designed primers to amplify the coding gene of phenazine-1-carboxylic acid biosynthesis, the product was detected by 0.7% agarose electrophoresis, and the length was recovered About 6.8kb gene fragment (phzA1-G1), the accuracy of the gene fragment w...

Embodiment 2

[0051] 1. The gene encoding phenazine-1-carboxylic acid biosynthesis was amplified by the same method as in Example 1. The product was detected by 1.0% agarose electrophoresis, and a gene fragment (phzA1-G1) with a length of about 6.8 kb was recovered.

[0052] 2. The recovered gene amplification fragment (phzA1-G1) was digested with restriction enzymes KpnI and BglII, ligated with ligase, and inserted into the E. coli / Pseudomonas shuttle expression plasmid pME6032, and phenazine-1 -Expression of genes encoding carboxylic acid biosynthesis is placed on strong promoter P tac Under control, the recombinant plasmid pME6032Phz was formed and transformed into E. coli. Finally, the recombinant plasmid was extracted from E. coli and verified.

[0053] 3. Prepare the competent cells of the growth-promoting antagonistic antibacterial M18 derivative strain M18G, and transform the above recombinant plasmid pME6032Phz into the competent cells of M18G, culture for 1 day at 30℃, and screen out t...

Embodiment 3

[0059] 1. The gene encoding phenazine-1-carboxylic acid biosynthesis was amplified by the same method as in Example 1. The product was detected by 1.0% agarose electrophoresis, and a gene fragment (phzA1-G1) with a length of about 6.8 kb was recovered.

[0060] 2. The recovered gene amplified fragment (phzA1-G1) was digested with restriction enzymes KpnI and BglII, ligated with ligase, and inserted into the E. coli / Pseudomonas shuttle expression plasmid pME6032, and phenazine-1 -Expression of carboxylic acid biosynthesis-encoding genes is placed on strong promoter P tac Under control, the recombinant plasmid pME6032Phz was formed and transformed into E. coli. Finally, the recombinant plasmid was extracted from E. coli and verified.

[0061] 3. Prepare the competent cells of the growth-promoting antagonistic antibacterial M18 derivative strain M18G, and transform the above recombinant plasmid pME6032Phz into the competent cells of M18G, culture at 32℃ for 2 days, and screen out the ...

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Abstract

The invention relates to a method for producing phenazine-1-carboxylic acid by using engineering bacterial strain M18G to carry plasmid pME6032Phz. The method is implemented by amplifying coding gene segment phzA1-G1 from cerebiogen antagonist antibiosis M18 genome for biosynthesis of phenazine-1-carboxylic acid, inserting the gene segment into expression plasmid pME6032, placing under control ofstrong promoten Ptac to construct a recombinant plasmid pME6032Phz; then introducing the recombinant plasmid in the derivative bacterial strain M18G of cerebiogen antagonist antibiosis M18 to construct an engineering bacterial strain M18G / pME6032Phz. The engineering bacterial strain can realize efficient and stable expression of coding genes while amplifying reproduction number of coding genes for biosynthesis of phenazine-1-carboxylic acid. Finally the engineering bacterial strain M18G / pME6032Phz is cultured in soybean meal culture solution to efficiently and stably produce phenazine-1-carboxylic acid with vastly enhanced yield. By using the high yield engineering bacterial strain to produce phenazine-1-carboxylic acid, the production cost can meet the agricultural requirement for application in preventing and curing rice sheath blight disease, and the invention is applicable to preparation of bactericide phenazino-1-carboxylic acid and can be used for preventing and curing plant diseases in large scale.

Description

Technical field [0001] The invention relates to a method for producing a microbial fungicide, in particular to a method for constructing a genetic engineering strain M18G / pME6032Phz carrying a plasmid pME6032Phz by using a growth-promoting antagonistic antibacterial derivative strain M18G to efficiently produce phenazine-1-carboxylic acid. It belongs to the technical field of microbial pesticide production. Background technique [0002] The loss caused by crop diseases accounts for about 25 to 75% of the total output. Taking rice, the main food crop in my country, for example, the annual planting area is about 400 million mu, with a yield of 400 kg per mu, and rice disease causes an average yield reduction of 10% every year. The economic losses amounted to more than 30 billion yuan, which seriously threatened the safe production of food crops. At present, in addition to the selection of improved varieties and improved cultivation measures, the control of plant diseases in product...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12R1/01C12N15/63
Inventor 许煜泉周泉
Owner SHANGHAI JIAO TONG UNIV
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