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Efficient Bt15A3 strain with excellent gene organization and its separation and application

A strain and gene technology, applied in application, genetic engineering, plant genetic improvement, etc., can solve the problems of inconvenient production of engineering bacteria and no abandonment of screening

Inactive Publication Date: 2004-09-01
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] It is precisely because of the inconvenience that engineering bacteria are used in production that people have not given up screening Bt strains with new cry genes or expected good gene combinations

Method used

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  • Efficient Bt15A3 strain with excellent gene organization and its separation and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0010] Example 1 Isolation and screening of Bt15A3 bacterial strain

[0011] (1) Weigh about 1 gram of soil sample into 10 ml of sterile water, mix well, take 1 ml into NB medium mixed with polymyxin and penicillin, and incubate at 30°C for 72 hours. The colonies similar to Bacillus thuringiensis were selected, and the smears were stained and examined under the microscope, and the colonies with high synchronous rate of spore crystal formation and polymorphic crystals such as diamond and square were further purified.

[0012] (2) Design specific primers based on the conserved regions of various cry genes, detect various cry genes on the purified Bt strains, and select strains with desired gene combinations.

[0013] (3) Inoculate the strains screened and the reference strains into the M1 fermentation medium respectively, the formula of which is: fish meal 3.0-3.5%, cornstarch 1.2-1.5%, beef extract 0.2-0.5%, inorganic salt 0.1-0.2% , pH7.5-8.0. Cultivate for 26-30 hours at 30°...

Embodiment 2

[0019] Example 2. The biological characteristics of the Bt15A3 strain and the detection of the cry gene

[0020] After 48 hours of culture with common NB slant, more than 95% of the cells formed spores and crystals. The 15A3 strain was determined by classical serological experimental method. The flagellar antigen of the strain belongs to H21 type, and it is Bacillus thuringiensis subsp.colmeri. spore morphology, but differs from the standard Kommer subsp.

[0021] The insecticidal crystal protein is encoded by six genes cryIAa, cryIAc, cryIC, cryID, cryII and cryV.

[0022] The spores are egg-shaped, and the bacterium flagellin antigens germinated by the spores belong to the H21 type, which is Bacillus thuringiensis Kommer subspecies, and the results of antibiotic spectrum determination: amp r 、polymy r 、str s 、erys 、tet s 、chl s .

[0023] The cry genes of the 15A3 strain and the H21 standard strain were detected by the PCR method with specific primers to prove that th...

Embodiment 3

[0025] Liquid fermentation production and products of embodiment 3.15A3 strain

[0026] (1) Inclined bacteria

[0027] NB medium slant: beef extract 0.5%, peptone 1.0%, NaCl 0.5%, agar powder 1.8%, pH7.2-7.4. After the culture medium is prepared, put it into test tubes or Keshiba bottles, sterilize it with high-pressure steam at 121°C for 30 minutes, take it out, place it on a slope, and place it in an incubator at 30°C for 24-48 hours, and then inoculate without the growth of miscellaneous bacteria: take preserved bacteria Inoculate a fresh slant with one loop, incubate at 30°C for 10-12 hours, observe under the microscope after staining the smear, the bacteria are thick rod-shaped, with blunt round ends, and the protoplasm is uniform without miscellaneous bacteria.

[0028] (2) Expansion culture of Kirschner flask

[0029] Put one ring of the above-mentioned activated bacteria into the liquid NB culture medium, shake and culture at 30°C 180rpm / min for 5-6 hours, take 2ml i...

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Abstract

A Bacillus thuringiesis 15A3 strain is disclosed, which has unique cry gene organization including crylAa, crylAc, crylc, crylD, coyII and cryV. It is obtained by screening with PCR method and bioactivity measuring method. Its wettable powder and liquid preparation has high effect on preventing and eliminating the pests, such as beet noctuid, cotton ballworm, etc..

Description

technical field [0001] The present invention relates to Bacillus thuringiensis subsp. Kommer 15A3 strain which is highly toxic to lepidopteran major pests beet armyworm and cotton bollworm. The strain (Bacillus thuringiensis subsp.colmer 15A3), referred to as Bt15A3, the gene encoding insecticidal crystal protein (ICP) carried by this strain and its excellent combination, and the highly efficient bacterial insecticide developed by this strain. Background technique [0002] Bt preparations are bacterial insecticides widely used in the world, accounting for 95% of the total biological insecticide output, and have a very important position in the practice of microbial control of pests (Yu Ziniu, etc. Bacillus thuringiensis Beijing Science Press 1990 ). The insecticidal activity of Bt comes from the ICP encoded by the cry gene carried by itself. Each ICP has a specific insecticidal spectrum. Among the ICP genes, cry1Aa, cry1Ab, and cry1Ac are the most toxic insecticidal agents,...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A01N63/00C12N1/20C12N15/32
Inventor 陈月华任改新王津红刘春勇冯书亮吴卫辉王飞李红秀
Owner NANKAI UNIV
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