Gene engineering bacteria for producing crown bacterin and its construction method

A technology of genetically engineered bacteria and coronatin, applied in the field of microorganisms, can solve the problems of difficulty in large-scale agricultural production and high production costs, and achieve the effect of solving low and unstable yields and increasing yields

Active Publication Date: 2008-06-25
CHENGDU NEWSUN CROPSCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the production cost is still relatively high, and it is difficult to put it into large-scale agricultural production for a while.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Pseudomonas syringae pv.glycinea as the starting strain was cultured on MG solid medium containing kanamycin (10 μg / mL) at 28°C for 48 hours; the cultured starting strain Inoculate in MG liquid medium (the final concentration of kanamycin is 10 μg / mL), and culture on a shaker at 28° C. and 260 rpm. When the OD value reaches about 0.7, centrifuge, discard the supernatant, wash the obtained cells with phosphate buffer solution with a pH value of 7.0 for 4-5 times, suspend the cells, and make the cell concentration 10 7 c.f.u.ml -1 ;

[0060] Use a 15W sterilizing ultraviolet lamp to irradiate the above-mentioned suspended bacterial liquid for 10 seconds at a distance of 33 cm; take 1.8 ml of the irradiated bacterial liquid, add 0.2 ml of 0.1 mg / ml NTG, and conduct a mutagenic reaction for 10 minutes; add 2 ml of pH5.6 Dilute the reaction solution with citric acid buffer to terminate the reaction, wash 2-3 times, centrifuge, and resuspend the precipitate with an equal vo...

Embodiment 2

[0063] Inoculate the new bacterial strain Pseudomonas syringae pathogenic strain CGMCC 1412 into MG liquid medium, cultivate it on a shaker at a temperature of 28°C and a rotation speed of 260rpm / min until the OD value reaches 0.7, and then inoculate according to the inoculation amount of 2%. Put it into a 500mL Erlenmeyer flask equipped with 100ml HSC liquid culture, ferment and culture on a shaker at a temperature of 18°C ​​and a speed of 280rpm / min for 7 days, extract coronatine, repeat three times, and the yields of coronatin are 90.1mg / L and 84.7mg / L respectively. mg / L, 92.4mg / L.

Embodiment 3

[0065] Inoculate the single bacterium colony of Pseudomonas syringae soybean pathogenic variety CGMCC 1412 of the present invention into a test tube equipped with MG liquid medium (concentration of kanamycin is 10 μg / mL), shake at a temperature of 28° C. and a rotating speed of 260 rpm / min. Cultivated on the bed for 36 hours, it is a first-class seed liquid;

[0066] 200ml MG liquid culture medium (concentration of kanamycin is 10μg / mL) is divided into three 500ml Erlenmeyer flasks, and the cultured first-grade seed liquid is inoculated in the above-mentioned three 500ml Erlenmeyer flasks according to 2% inoculation amount , cultivated on a shaker at a temperature of 28°C and a rotation speed of 260rpm / min for 48 hours, which is a secondary seed solution;

[0067] Put 5L of HSC medium in a 7L fermenter, sterilize it with conventional high-pressure hot steam, inoculate the secondary seed liquid with 2% inoculum, and aerate and stir; the fermentation temperature is 18°C, and the...

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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

technical field [0001] The invention belongs to the technical field of microorganisms, and in particular relates to a genetically engineered bacterium for producing coronatin and a construction method thereof. Background technique [0002] Coronatine C 18 h 25 NO 4 , with a molecular weight of 319 (Ichihara et al, 1977), composed of two components, a coronamic acid containing an amino acid and a coronafacic acid with a polyketide structure, linked by amide bonds (Mitchell et al , 1994). It was first reported in the literature as a phytotoxin. However, recent studies have found that coronatin has similar functions to the plant growth regulator substances abscisic acid and jasmonic acid, and its activity is thousands of times that of the latter two. substitution. Moreover, coronatin is a pure natural product, and its application in production will not cause pollution to the environment. It is an environmentally friendly biological regulator. [0003] There are two metho...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12N13/00
Inventor 李召虎章家长段留生张明才田晓莉王保民焦睿吴惠玲
Owner CHENGDU NEWSUN CROPSCI
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