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Bacillus subtilis engineering bacteria resistant to rice white-backed planthopper, preparation method and application

A technology of Bacillus subtilis and engineering bacteria, which is applied in the fields of botany equipment and methods, microorganism-based methods, biochemical equipment and methods, etc., and can solve problems such as difficult operation of transgenic technology

Inactive Publication Date: 2011-12-14
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although transgenic plants have been used in the variety improvement of various crops, there are still some disadvantages, such as: transgene silencing, gene drift, some plant tissue culture techniques and transgenic techniques are difficult to operate, such as rice, cotton, etc.

Method used

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  • Bacillus subtilis engineering bacteria resistant to rice white-backed planthopper, preparation method and application
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  • Bacillus subtilis engineering bacteria resistant to rice white-backed planthopper, preparation method and application

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Experimental program
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Effect test

Embodiment 1

[0055] A preparation method of rice endophyte Bacillus subtilis, the steps are:

[0056] 1. Screening and isolation of Bacillus subtilis:

[0057] Take Zhonghua 11 healthy rice at the tillering stage from the paddy field of Huazhong Agricultural University in Wuhan City, Hubei Province, China, fully wash the surface soil with tap water, cut into 2cm×2cm or 4cm small pieces, and rinse with 75% (v / v) ethanol for 5min , adding 0.2% HgCl 2 (v / v) Disinfect the surface for 5 minutes, and then rinse it with sterile water for 5 times. Under sterile conditions, the tissue was thoroughly ground, and then centrifuged at 2000 g for 5 min. Take the supernatant and smear LB medium (containing 10g peptone, 5g yeast powder, 10g NaCl, 20g agar, pH 7.0-7.2, add distilled water to 1L) and incubate at 37°C for 48h, pick a single bacterial colony, see reference (Misaghi, I.J., Donndelinger, C.R., Endophytic bacteria in symptom-free cotton plants. Phytopatho, 1990, 80:808-811).

[0058] 2. Clas...

Embodiment 2

[0075] A kind of Bacillus subtilis engineering bacterium strain WH2 is used in the preparation treatment or prevents the application of rice white-backed planthopper medicament, and its specific steps are:

[0076] 1. Colonization separation and colonization range determination:

[0077] Inoculate Bacillus subtilis engineered bacteria WH2 in 30ml LB and shake culture for 24h, centrifuge at 8 000r / min for 5min, discard the supernatant, suspend the bacteria with sterile water, and prepare the bacteria with a concentration of 8×10 6 CFU / ml of bacterial liquid.

[0078] Rice seeds were soaked in sterile water at room temperature (22-25°C) for 30 hours, soaked in a 1 000-fold diluted carbendazim solution at 37°C for 7 hours, sterilized with 75% (v / v) alcohol for 1 minute, and finally washed with 1 %(v / v) mercuric chloride was treated for 3 minutes and washed with sterile water, sowed on PSA medium (containing 200g potatoes, 20g sucrose, distilled water to 1L), cultured in a light ...

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Abstract

The invention discloses a Bacillus subtilis engineering bacterium resistant to rice white-backed planthopper and its preparation method and application. The steps are: A. screening and isolation of Bacillus subtilis; B. classification and identification of bacteria: conducting 16SrRNA and Morphological identification; C. Construction of a recombinant vector expressing Pinellia lectin: using Pinellia lectin to construct a secretory expression shuttle vector, pP43NMK and Pinellia lectin encoding genes were digested with PstⅠ and HindⅢ double enzymes to construct a recombinant vector containing Pinellia lectin A vector of a lectin-encoding gene; D. Construction of Bacillus subtilis engineering strain WH2: the constructed pP43NMK-pta plasmid was transformed into Bacillus subtilis by Spizizen method. It has obvious anti-insect activity, the method is simple, Bacillus subtilis can stably colonize in plants, and the recombinant bacteria Bacillus subtilis has obvious anti-white-backed planthopper activity; LB medium, storage: Take the cultured Bacillus subtilis WH2 bacterial liquid and add glycerol to the final concentration for storage.

Description

technical field [0001] The invention belongs to the technical field of agricultural microbiology, and specifically relates to a rice endogenous Bacillus subtilis, and also relates to a preparation method of a Bacillus subtilis engineering bacterium, and also relates to a Bacillus subtilis engineering bacterium resistant to rice white-backed planthoppers the use of. Background technique [0002] Rice planthopper is the most important pest of rice in my country, and has formed a serious threat to the current rice production in my country. Rice planthoppers are homopteran pests that suck the sap of rice. Due to the damage of the transporting tissue of the damaged plants, the nutrients cannot be sent normally, which affects the growth and development, resulting in thin and weak stems, short plants, unfulfilled grains, and gradually withered seedlings. to death. In addition, rice planthoppers produce wounds when piercing through their mouthparts, and transmit various viruses th...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/75A01N63/00A01P7/04C12R1/125
Inventor 赵秀云祁高富张献芳张翼王圣英
Owner HUAZHONG AGRI UNIV
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