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Efficient thuricin bacillus strain for killing lepidopteron, its insecticide gene and use

A technology of Bacillus chrysogenum and Lepidoptera, which can be applied in further fields, can solve problems such as destruction of ecological balance, survival and health hazards, and increase in pesticide residues

Inactive Publication Date: 2007-11-21
李平 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In order to reduce these losses, chemical prevention and control methods are generally used for prevention and control. However, due to the long-term and large-scale use of chemical pesticides, the pollution of the environment has been caused, and the amount of pesticide residues in agricultural and sideline products has increased, which has brought harm to human survival and health.
In addition, while chemical pesticides kill pests, they also kill natural enemies and other beneficials, destroying the ecological balance

Method used

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  • Efficient thuricin bacillus strain for killing lepidopteron, its insecticide gene and use
  • Efficient thuricin bacillus strain for killing lepidopteron, its insecticide gene and use
  • Efficient thuricin bacillus strain for killing lepidopteron, its insecticide gene and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Example 1 Screening and identification of Bacillus thuringiensis containing multiple cry genes

[0072] The soil was collected from the Wenjiang area of ​​Chengdu, Sichuan Province. Adopt sodium acetate-antibiotic separation method, take 10g soil sample and put into the shaking flask that 50ml sodium acetate medium is housed, add respectively 400 μ g / ml of penicillin sodium salt and gentamicin sulfate, shake table culture (200r / 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 Figure 1). Observed by optical microscope and electron...

Embodiment 2

[0073] Example 2 Identification of cry gene in bacterial strain Rpp02

[0074] Design several pairs of specific primers based on the conserved sequence of the cry1 gene:

[0075] cry1Ab: 5'-CGCTAACGCAATTTCTTTTGAGTG-3'

[0076] 5'-GAGCCAAGATTAGTAGATTTTGTTAA-3'

[0077] cry1Ac: 5'ATCACTGAGTCGGTTCGCATGTTTGACTTTATC-3'

[0078] 5'-TCACTTCCCATCGACATCTACC-3'

[0079] cry1C: 5'-CCACAGTTACACTCTGTAGCTC-3'

[0080] 5'-CACTTAATCCTGTGACGCCT-3'

[0081] Design a pair of universal primers based on the cryV gene:

[0082] cryV: 5'-ATGAAACTAAAGAATCAAGA-3'

[0083] 5'-ACCTGTGCTATACAATTTCA-3'

[0084] Use the following PCR reaction system for identification:

[0085] 10×buffer 2.5ul

[0086] MgCl 2 (25mM) 1.5ul

[0087] Taq enzyme 0.2ul

[0088] dNTPs (2.5mM) 2ul

[0089] Primer 2ul

[0090] Template 5ul

[0091] Final reaction volume 25ul

[0092] Thermal cycle reaction: pre-denaturation at 94°C for 5 minutes; denaturation at 94°C for 1 minute; anneali...

Embodiment 3

[0093] Example 3 Cloning of cry1Ac gene in bacterial strain Rpp02

[0094] Genomic DNA Purification Kit (purchased from Saibaisheng Company) was used to extract the total DNA of strain Rpp02, and according to the sequence of cry1Ac in Genbank, primers P1 and P2 of its full-length gene were designed (primer sequences are as follows), and the total DNA of strain Rpp02 was used as a template , amplify the full-length cry1Ac gene to obtain a fragment of about 4k in length, connect the purified PCR product to the pGEM-T vector, transform, and screen a positive clone pGF10 (see Figure 4), and sequence to obtain SEQ ID NO1, the full length of the sequence is 3734bp, analysis shows that it contains a large open reading frame ORF, its position is 181-3714, GC content is 39.3%, and it encodes a protein consisting of 1177 amino acids. After determination, the composition of its amino acid sequence is shown in SEQ ID NO2. Using the bacteria sigma7.0 promoter program on the softberry webs...

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Abstract

An efficient Rpp02 for killing lepidopteron, its gene for killing insects and use, cry1AC gene nucleotide sequence, its encoded protein amino acid sequence, shuttle expression carrier pCA-1AC and its construction are disclosed. The Rpp02 contains cry1Ac, cry1Ab, cry1C and crylV etc. multi-genes. It has high toxicity for lepidopteron and delays drug resistance for engineering bacterium and transfer-gene plant.

Description

technical field [0001] The invention belongs to the technical field of biological control. Specifically, the invention relates to a Bacillus thuringiensis strain Rpp02 containing multiple cry genes, which has at least an excellent combination of cry1Ac, cry1Ab, cry1C and cryV important insecticidal genes, and is beneficial to agricultural production. Some of the main pests, especially lepidopteran pests such as vegetables, cotton, corn, rice, and forests, have relatively high activity. Further, the present invention relates to the nucleotide sequence of Bt cry1Ac gene and its coded protein amino acid sequence, which are highly toxic to Lepidoptera, and to the shuttle expression vector pCA-1Ac expressed in plants. Background technique [0002] In the process of agricultural production, insect pests are an important factor causing agricultural production losses. According to the statistics of FAO, the annual economic losses caused by insect pests in the world's agricultural pr...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/32C07K14/325A01N63/00A01P7/04C12R1/07
Inventor 李平谭芙蓉郑爱萍
Owner 李平
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