CN102876597A

CN102876597A - Preparation and application of ribonucleic acid (RNA) polymerase mutant for highly yielding antifungal substance Iturin A

Info

Publication number: CN102876597A
Application number: CN2011103989642A
Authority: CN
Inventors: 刘常宏
Original assignee: Individual
Current assignee: Individual
Priority date: 2011-12-06
Filing date: 2011-12-06
Publication date: 2013-01-16

Abstract

The invention belongs to the technical field of biopesticides and particularly relates to a ribonucleic acid (RNA) polymerase mutant, which is screened by a rifampicin-containing (50mu g/ml) Luria-Bertani (LB) plate and used for highly yielding Iturin A, of an Acinetobacter baumannii LCH0606 strain. Mutational sites of the mutant are positioned at the 1,619th basic group, the 1,603th basic group and the 1,604th basic group of an rpoB gene for encoding a beta sub-unit of RNA polymerase. Compared with a wild type LCH0606 strain, the mutant has the advantages that the ability of producing the antifungal substance Iturin A is improved by 95 to 300 percent, and the antifungal activity is improved by 20 to 70 percent; and the mutant can be used for the control of various crop diseases and continuous cropping obstacles of medicinal plants.

Description

The preparation and application of the RNA polymerase mutant of a kind of high yield antifungal substance Iturin A

Technical field

The invention belongs to biological pesticide technical field, be specifically related to screening and the application thereof of RNA polymerase mutant of the Acinetobacter baumannii LCH0606 bacterial strain of a kind of high yield antifungal substance Iturin A.

Background technology

All crises that the raising that quality of life is required along with people and environmental ecology face; the ultimate challenge that agriculture production faces is exactly how can find the substitute of environmentally friendly chemical pesticide to resist agricultural disease in future; along with various countries scientist's research is found; coming from the beneficial microorganism biological pesticide is substitute (the Marc Ongena etc. that promise to be this series products most; Cell; 2007,16 (3): 115-125).Microorganism is biological group the hugest on the earth on the one hand, it not only has widely species diversity, but also can increase substantially by strain improvement, metabolic regulation, scale fermentation and other modern biotechnology the output of target substance, be easy to realize suitability for industrialized production; On the other hand, microbial bactericide (microorganism and the meta-bolites thereof of living) is safer than chemical pesticide, easily degraded, and synthetic cost is lower.Wherein produced by U.S. Agraquest company based on the biological pesticide of Bacillus subtilis QST713 and QST2808 bacterial strain, and commodity SerenadeTM by name and Sonata AS (Cao CX etc., Agric.Nat.Resour, 2010, SAG-18-10).

Yet, because active component content is lower in the microbial pesticide, seriously restricted production and the popularization of microbial pesticide.The method that is used at present raising microbial pesticide active substance mainly contains:

Optimal Medium forms and fermentation condition (Zmijewski M J etc., J Antibiot, 1986,39 (10): 1483);

Derivatization treatment (Boeck L D etc., J Antibiot, 1990,43 (6): 607) of precursor control and product; The screening of excellent species and selection by mutation (Li Jing etc., Agriculture of Anhui science, 2008,36 (1): 106-111,132).

Core mutant of the present invention derives from Acinetobacter baumannii LCH0606 (Acinetobacter baumannii LCH0606; CGMCC No.1752) and (license number: ZL200610088339.7) of biocontrol microorganisms patented product culture presevation number:, the antimycotic mechanism of this biocontrol fungicide is to synthesize broad-spectrum antifungal material Iturin A, it has significant prevention effect to diseases such as soybean anthracnose, Phytophthora capsici disease, graw mold of tomato, wheat scab and rice sheath blight diseases, especially medicinal plant glutinous rehmannia continuous cropping obstacle is had good control action kou.For this reason, the present invention by RNA polymerase spontaneous mutation technology, has obtained the RNA polymerase mutant of high yield Iturin A take this biocontrol microorganisms as the basis, for the industrialization production of Iturin A provides a new approach and produced bacterial strain.

Summary of the invention

Main purpose of the present invention is: a kind of technology of utilizing the RNA polymerase mutating technology to improve Iturin A is provided, the RNA polymerase mutant of the Acinetobacter baumannii LCH0606 of the Iturin A output increased that obtains, and be applied to the control of Plant diseases and medicinal plant continuous cropping obstacle.

Technical scheme of the present invention:

1.LCH0606 the acquisition of bacterial strain RNA polymerase mutant

The activation of bacterial classification

Bacterial strain LCH0606 is activated in the LB liquid nutrient medium, then be inoculated in 8 test tubes that contain the LB liquid nutrient medium and further cultivate.

Dull and stereotyped the producing of mutant bacteria screening

After LB solid medium heating and melting, add certain density Rifampin solution in the substratum that has melted, under aseptic condition, fall flat board in diameter as 90mm take the amount of 20-30ml after shaking up.

Will

In 8 original strain seed liquor, evenly coat 8 with the inoculum size of every dull and stereotyped 200 μ L respectively

On the flat board that method makes.37 ℃ of dark places are cultivated and were taken out afterwards in 2 days, the finding bacterium colony namely might be the RNA polymerase mutant, and then by the sequential analysis to PCR and the PCR product of rpoB gene in this mutant, determine further whether the mutator gene of mutant occurs on the rpoB gene of coding RNA polymerase beta subunit.

2. mutant and produce the assay of Iturin A

By suppressing plant pathogenic fungi activity experiment and HPLC check, determine the anti-mycotic activity of High yield Mutant and the content of the Iturin A that produces.

3. the High yield Mutant mutant gene locus determines

Utilize DNA of bacteria to extract test kit, extract the DNA of a mutant, and carry out pcr amplification with a series of primers, obtain complete rpoB gene order, through sequential analysis and with the sequence analysis of wild type strain rpoB sequence, find out mutating alkali yl and corresponding amino acid.

4. the Iturin A of mutant and generation thereof is to the control experiment of plant pathogenic fungi

Adopt experiment in the ware, measure each mutant fermented liquid and bacteria-free filtrate anti-plant pathogenic fungi growth activity thereof.

5. the effect of mutant in control glutinous rehmannia continuous cropping obstacle

Adopt the seed soaking mode, the effect of research LCH0606 bacterial strain mutant in control glutinous rehmannia continuous cropping obstacle.Seed soaking time is 30 minutes.

The present invention's beneficial effect compared with prior art:

[1] the Iturin A that the mutant that obtains produces significantly improves (95%～300%) than wild strain, and antibiotic generation time in advance, and anti-mycotic activity improves 20%～70% than wild-type LCH0606 bacterial strain.

[2] without crossing the people for inducing, the gained mutant comes from the spontaneous mutation of wild type strain, and physio-biochemical characteristics and the inherited character of mutant are relatively stable.

[3] mutant that obtains and the Iturin A that produces thereof can be applicable to prevent and treat soybean anthracnose (Glomerella glycines), wheat scab (Fusarium graminearum), rice sheath blight disease (Rizoctonia solani), interlinkage spore leaf spot (Alternaria alternata), Phytophthora capsici sick (Phytophthora capsici) and medicinal plant glutinous rehmannia continuous cropping obstacle.

Description of drawings

Fig. 1. wild-type Acinetobacter baumannii LCH0606 (WT) and the restraining effect of its RNA polymerase mutant to plant pathogenic fungi interlinkage spore (Alternaria alternata).

Fig. 2. the comparison that wild-type Acinetobacter baumannii LCH0606 (WT) and its RNA polymerase mutant are produced IturinA.

Fig. 3. the disease severity behind wild-type Acinetobacter baumannii LCH0606 (WT) and its RNA polymerase mutant (Abm03) control medicinal plant glutinous rehmannia continuous cropping obstacle.

Fig. 4. the effect of increasing production behind wild-type Acinetobacter baumannii LCH0606 (WT) and its RNA polymerase mutant (Abm03) control medicinal plant glutinous rehmannia continuous cropping obstacle.

Embodiment

1.RNA the screening of polymerase mutant

The activation of bacterial classification

Wild-type Acinetobacter baumannii LCH0606 (WT) and its RNA polymerase mutant production are inoculated in the LB liquid nutrient medium of 4mL/ pipe with 1% inoculum size, 37 ℃, 120rpm activation culture 48h, then carry out re-activation in 8 LB liquid nutrient mediums with identical inoculum size and culture condition, cultivate 24h.

Contain the preparation of Rifampin flat board

Rifampin solution added in 50～60 ℃ the LB solid medium, final concentration is 50 μ g/mL, is to contain the Rifampin flat board.

Will

The re-activation seed liquor of middle preparation (8 test tubes) is evenly coated respectively by the amount of 200 μ L/ wares and to be contained on the Rifampin flat board, and 37 ℃ of dark places are cultivated after 2 days and observed bacterium colony, are possible RNA polymerase mutant, sieve altogether to get 78 plant mutant bodies.

2. High yield Mutant and Iturin A content thereof determines

Suppress the plant pathogenic fungi activity experiment

Take the interlinkage spore as the test plants pathogenic fungi.Fermented liquid is behind the centrifugal removal cell of 12000rpm, 10min, the aseptic supernatant liquor 1ml of gained mixes with 10mL PSA substratum and pours culture dish (9 centimetres of diameters) into, the fresh test fungi bacterium cake (diameter 7mm) of inoculation after the cooling, bacterium cake back-off is in each culture dish, cultivate after 48 hours for 28 ℃ and measure colony diameter, calculate growth inhibition ratio.Growth inhibition ratio (%)=[(the bacterial plaque diameter of negative control bacterial plaque diameter-disposable plates)/(negative control bacterial plaque diameter-bacterium cake diameter)] * 100%.

Result's (seeing Fig. 1) demonstration, the anti-mycotic activity of mutant Abm03, Abm05 and Abm28 improves 20%～70% than wild type strain.

The HPLC of Iturin A output detects

Sample preparation: quantitative Abm03, Abm05 and Abm28 fermented liquid, after the centrifugal acquisition supernatant liquor, for the detection of Iturin A, testing conditions is: moving phase: A pump 5mM ammonium acetate (chromatographically pure), B pump acetonitrile, A: B are 55: 45; Flow velocity: 1 ml/min; Detector: 220nm.

Result (Fig. 2) demonstration, the Iturin A that mutant Abm03, Abm05 and Abm28 produce improves 95%～300% than wild type strain.

3. the mutant mutant gene locus determines

The centrifugal acquisition thalline of mutant Abm03, Abm05 and Abm28 fermented liquid adopts DNA of bacteria to extract test kit and extracts genomic dna, and according to homology rpoB primers among the NCBI, gained DNA is through pcr amplification.The flow process of PCR reaction is: 94 ℃ of denaturations 5 minutes; 30 circulations, comprising: 94 ℃ of sex change 30 seconds, 55 ℃ of annealing 1min, 72 ℃ are extended 1min; After reaction finishes, in 72 ℃ of downward-extensions 7 minutes.Gained PCR product is delivered the order-checking of the large genome company of China.With wild type strain rpoB Gene sequence comparison, mutant Abm03, the mutational site of Abm05 and Abm28 betides respectively on the rpoB gene of coding RNA polymerase beta subunit, wherein the mutational site of mutant Abm03 occurs in the 1619th base, C sports T by base, cause amino acid to become phenylalanine (F) by Serine (S), the sudden change of mutant Ab05 occurs in the 1603rd base, C sports T by base, cause amino acid to become halfcystine (C) by Histidine (H), the sudden change of mutant Ab28 occurs in the 1604bp base, A sports G by base, causes amino acid to become arginine (R) by Histidine (H).

The fermented liquid of mutant and the bacteria-free filtrate of centrifugal gained (being Iturin A solution) thereof are carried out respectively the anti-plant pathogenic fungi growth experiment, the result is as shown in table 1, mutant and the Iturin A that produces thereof are to soybean anthracnose (Glomerella glycines), Phytophthora capsici sick (Phytophthora capsici), wheat scab (Fusarium graminearum), rice sheath blight disease (Rizoctonia solani), interlinkage spore leaf spot (Alternaria alternata) all has good preventive and therapeutic effect.

Table 1. mutant Abm03 is to the prevention effect (%) of plant pathogenic fungi

With mutant Abm03 and wild type strain LCH0606 seed soaking glutinous rehmannia piece root, by normal cultivation step, when gathering in the crops, glutinous rehmannia investigates its disease severity and output, find the disease severity difference 21.0% and 18.6% with the plant of LCH0606 bacterial strain and mutant strain Abm03 processing thereof, with prevention effect (17.4%) there was no significant difference of positive control derosal (Carbendazim), and significantly be lower than water (H ₂O) seed soaking is negative processes (37.4%) (Fig. 3).

Wild-type LCH0606 and mutant Abm03 thereof not only have higher preventive effect to Herba Munroniae henryi as obstacle, and can improve glutinous rehmannia output, wherein LCH0606 and mutant Abm03 thereof process respectively and increase production 28.6% and 33.7% than negative control, are significantly higher than the volume increase level (27.1%) of positive control derosal processing (Fig. 4).

Claims

1. the RNA polymerase mutant of a high yield antifungal substance Iturin A, it is characterized in that producing the endophyte of plant of Iturin A---Acinetobacter baumannii LCH0606 is the RNA polymerase mutant of this bacterium of Rifampin LB plate screening of 50 μ g/ml for the basis with concentration.The anti-mycotic activity of this mutant improves 20%～70% than wild-type, and antifungus active substance Iturin A output improves 95%～300% than wild type strain.

2. the mutational site of the RNA polymerase mutant of high yield antifungal substance Iturin A according to claim 1, it is characterized in that the mutational site is positioned at the rpoB gene order of coding RNA polymerase beta subunit, wherein the mutational site of mutant Abm03 occurs in the 1619th base, C sports T by base, cause amino acid to become phenylalanine (F) by Serine (S), the sudden change of mutant Ab05 occurs in the 1603rd base, C sports T by base, cause amino acid to become halfcystine (C) by Histidine (H), the sudden change of mutant Ab28 occurs in the 1604bp base, A sports G by base, causes amino acid to become arginine (R) by Histidine (H).

3. the preparation method of the RNA polymerase mutant of high yield antifungal substance Iturin A according to claim 1, it is characterized in that wild type strain LCH0606 is inoculated in the sterilising liq LB substratum of pH7.0, evenly coat the LB solid plate that contains Rifampin 50 μ g/ml after cultivating 24h, the bacterium colony that grows after cultivate-fixing time is the RNA polymerase mutant of wild type strain LCH0606.

4. the RNA polymerase mutant of high yield antifungal substance Iturin A according to claim 1 is produced the method for Iturin A, it is characterized in that this mutant is inoculated in liquid LB substratum with 1% inoculum size, 37 ℃, 120rpm cultivates 48h, the antimycotic Iturin A of fermentative production.

5. the RNA polymerase sudden change that utilizes described in according to claim 1 improves the mutant of Iturin A output in control soybean anthracnose (Glomerella glycines), wheat scab (Fusarium graminearum), rice sheath blight disease (Rizoctonia solani), the application in interlinkage spore leaf spot (Alternaria alternata), Phytophthora capsici sick (Phytophthora capsici) and the medicinal plant continuous cropping obstacle.

6. the microbiotic that produces of the mutant that utilizes the RNA polymerase sudden change to improve Iturin A output described in according to claim 4 is in control soybean anthracnose (Glomerella glycines), wheat scab (Fusarium graminearum), rice sheath blight disease (Rizoctonia solani), the application in interlinkage spore leaf spot (Alternaria alternata), Phytophthora capsici sick (Phytophthora capsiici) and the medicinal plant continuous cropping obstacle.