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New strain of bacillus thuringiensis and application thereof

A technology of Bacillus thuringiensis and bacteria agent, applied in the field of Bacillus thuringiensis and its application in agricultural pest control, can solve the problem of undiscovered resistance and other problems

Active Publication Date: 2016-03-02
四川新思原高科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, studies have shown that resistance problems have not been found in the use of Bti in field (RegisL, et al., 2000. The use of bacterial larvicides in mosquito and blackfly control programs in Brazil. Mem. Instituto Oswaldo Cruz, 95:207-210.), but the problem of mosquito resistance to it has been confirmed in the laboratory, and this situation may also appear in the field (GeorghiouGP, and WirthMC, 1997.Influence of exposure to singleversusmultipletoxinsof Bacillus thuringiensis subsp. israelensis ondevelopmentofresistanceinthemosquito Culexquinquefasciatus (Diptera: Culicidae). Applied and Environmental Microbiology, 63:1095-1101.)

Method used

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  • New strain of bacillus thuringiensis and application thereof
  • New strain of bacillus thuringiensis and application thereof
  • New strain of bacillus thuringiensis and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1 contains a variety of cry Screening and Identification of Genes of Bacillus thuringiensis

[0022] The soil was collected from the soil in Chengdu City, Sichuan Province. Using the sodium acetate-antibiotic separation method, weigh 10 g of soil samples and put them into a shaker flask filled with 50 ml of sodium acetate medium, add penicillin sodium salt and gentamicin sulfate at 400 μg / ml each, and culture on a shaking table (200 r / min , 30°C) 4h. After the cultivation, take 10ml of soil suspension, put it into a sterile centrifuge tube and centrifuge at 3000r / min for 15min, take 2ml of the upper cloudy solution and place it in a water bath at 65°C for 15min, take 0.1ml of the heat-treated cloudy solution and spread it on a plate, and place the plate in a 30°C incubator cultivated in. After 48 hours, smears of Bt-like strains were picked from the plate. A Bt strain with rhombohedral crystal morphology was found (see attached figure 1 ). Observed by op...

Embodiment 2

[0023] In Example 2 bacterial strain BN23-5 cry Identification of genes

[0024] according to cry Design a pair of specific primers for the conserved sequence of class 1 genes:

[0025] K5un2 (SEQ ID NO1): AGGACCAGGATTTACAGGAGG

[0026] K3un2 (SEQ ID NO2): GCTGTGACACGAAGGATATAGCCAC

[0027] Use the following PCR reaction system for identification:

[0028] 10×buffer2.5μl

[0029] MgCl 2 (25mM) 1.5μl

[0030] Taq enzyme 0.2μl

[0031] dNTPs (2.5mM) 2μl

[0032] Primer 2μl

[0033] Template 5 μl μ

[0034] Final reaction volume 25 μl

[0035] Thermal cycle reaction: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 1 min, annealing temperature depends on the primer, extension at 72°C for 2 min, 30 cycles; extension at 72°C for 5 min; stop reaction at 4°C. The amplification reaction products were electrophoresed on 1% agarose gel, and the PCR amplification results were observed in a gel imaging system. The result is as image 3 As shown, the above prime...

Embodiment 3c

[0036] Example 3 cry1Ha-like gene cloning

[0037] Genomic DNA purification kit (purchased from Saibaisheng Company) was used to extract the total DNA of strain BN23-5; design its full-length gene primers P1 and P2 (primer sequences are as follows); Primers carry out PCR amplification, and reaction system and reaction procedure are with embodiment 2; Amplify with primer P1 and P2 cry1Ha-like The full-length gene obtained a 3507bp fragment (see attached Figure 4 ). The purified PCR product was connected to the pGEM-T vector, transformed, and positive clones containing the target fragment were picked and sequenced to obtain the sequence SEQIDNo.1 respectively.

[0038] P1 (SEQ ID NO3): 5'ATGGAGAATAAAAATCAACAC3'

[0039] P2 (SEQ ID NO4): 5'CTATTCCTCCATAAGGAG3'

[0040] cry1Ha-like gene sequence analysis

[0041] The full length of the sequence SEQIDNo.7 is 3507bp, the analysis shows that the GC content is 39.58%, and it encodes a protein consisting of 1169 amino acids....

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PUM

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Abstract

The invention provides a new strain BN23-5 of bacillus thuringiensis. The preservation number is CGMCCNO.9448. By testing the activity of virulence of the BN23-5, it shows that the BN23-5 has extremely high virulence on lepidoptera and the like. The BN23-5 of bacillus thuringiensis can be prepared into insecticide and is used for preventing and treating important crop pests. Thus, bacillus thuringiensis insecticide products can be diversified and serialized, the using range of bacillus thuringiensis insecticide is widened, the using amount of pesticide is reduced, environmental pollution is reduced, and the new strain BN23-5 of bacillus thuringiensis has important economic value and application prospects.

Description

technical field [0001] The invention relates to a new microbial strain and its application, in particular to a bacillus thuringiensis and its application in agricultural pest control. Background technique [0002] In the process of human production, insect pests are an important factor that causes agricultural production losses and affects human health. According to the statistics of FAO, the annual economic loss caused by insect pests in the world's agricultural production is as high as 14%, the loss of diseases is 12%, and the loss of weeds is 11%. The loss was as high as 126 billion US dollars, equivalent to half of China's total agricultural output value, and more than four times that of the United Kingdom. In order to reduce these losses, for many years, chemical control methods have been widely used to control crop pests and mosquitoes. However, due to the long-term and large-scale use of chemical pesticides, the pollution to the environment has been caused, and the a...

Claims

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Application Information

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IPC IPC(8): C12N1/20A01H5/00A01N63/00A01N63/02A01P7/04C12R1/07
Inventor 郑爱萍余宗兰陈磊王娜李巧李平王玲霞刘怀年李双成朱军邓其明王世全
Owner 四川新思原高科技有限公司
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