Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Bacillus thuringiensis cry1Ca gene, expressed protein and application of bacillus thuringiensis cry1Ca gene

A Bacillus thuringiensis and genetic technology, applied in the fields of application, genetic engineering, plant genetic improvement, etc., can solve the problems of increasing insecticide resistance and singleness of pests, and achieve the effects of expanding insect spectrum, wide application prospects, and delaying insecticide resistance

Inactive Publication Date: 2012-07-11
NORTHEAST AGRICULTURAL UNIVERSITY
View PDF4 Cites 11 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Since the current commercialized transgenic insect-resistant crops have relatively single types of insect-resistant genes, such large-scale promotion of planting has the risk of reducing pest refuges and increasing pest resistance.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Bacillus thuringiensis cry1Ca gene, expressed protein and application of bacillus thuringiensis cry1Ca gene
  • Bacillus thuringiensis cry1Ca gene, expressed protein and application of bacillus thuringiensis cry1Ca gene
  • Bacillus thuringiensis cry1Ca gene, expressed protein and application of bacillus thuringiensis cry1Ca gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1, isolate Bacillus thuringiensis bacterial strain LB-R-78

[0037]The applicant's laboratory staff obtained a strain of Bacillus thuringiensis from the soil near Lalin Town, Wuchang City, Heilongjiang Province. membranes and protoplasts. The main component of the cortex is peptidoglycan, a polysaccharide teichoic acid that does not contain vegetative cells, which maintains the dehydration state and heat resistance of the spores. On the other hand, during the formation of the spores, a large amount of DPA-Ca will be produced. thuringiensis spores will not die after heat treatment at 80°C for 20 minutes, and the dormant spores are treated at a sub-lethal temperature of 75°C for 15 minutes, the activation effect is the best , not only promote its rapid germination, but also improve the survival rate of spores (Yu Ziniu 1990). According to this characteristic, temperature screening can be implemented (Knowles B H, Ellar D J. Colloid-osmotic lysis is a general ...

Embodiment 2

[0053] Example 2. Obtaining new genes

[0054] 2.1 Use cry1 gene universal primers to detect strain LB-R-78, the primers are as follows

[0055]

[0056]

[0057] Amplification cycle: denaturation at 94°C for 1 minute, annealing at 56°C for 1 minute, extension at 72°C for 4 minutes, 25 cycles, and finally extension at 72°C for 10 minutes.

[0058] The result is as image 3 As shown, strain LB-R-78 was identified by PCR-RFLP genotype, and a PCR product with a size of 1.6 kb was obtained by using cry1 gene identification primer K5un2 / K3un2, which was subjected to Pst I / Xba I double enzyme digestion analysis, The RFLP map was obtained. It can be seen from this figure that the amplified 1.6kb PCR product produced 9 restriction fragments after enzyme digestion, the sizes were 801bp, 758bp, 518bp, 423bp, 322bp, 239bp, 140bp, 95bp, 16bp (Kuo et al, 1996), the results showed that: LB-R-78 contains cry1Ca and cry1Ac genes.

[0059] 2. Cloning of cry1Ca gene in 1LB-R-78 strain ...

Embodiment 3

[0107] Embodiment 3, gene expression and activity assay

[0108] 3.1.1 Plasmid DNA was extracted from the above clones, and transformed into the recipient strain Rosetta (DE3) to obtain an expression strain.

[0109] After IPTG induced expression, SDS-PAGE protein electrophoresis was performed.

[0110] The process of inducing expression is as follows:

[0111] 1) Activated strains (37°C, 12hr);

[0112] 2) 10% inoculated in LB medium (37°C, 2hr);

[0113] 3) Add the inducer IPTG, 150rpm, and induce at 18-22°C for 4-20h at low temperature;

[0114] 4) The cells were collected by centrifugation, and 10 mM Tris Cl (pH 8.0) was added to suspend;

[0115] 5) Broken bacteria (ultrasonic crushing is complete);

[0116] Centrifuge at 12,000rpm for 10min at 4°C;

[0117] Collect 10-15 μL each of the supernatant and the precipitate, and detect them by electrophoresis.

[0118] The polyacrylamide gel configuration is as follows.

[0119]

[0120] Sample loading: 10-15μl sampl...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention relates to a bacillus thuringiensis cry1Ca gene, expressed protein and application of the bacillus thuringiensis cry1Ca gene and belongs to the technical field of biological prevention and control. Insecticidal protein has amino acid sequence shown as SEQ ID NO.2. In a gene for encoding the insecticidal protein, preferably, the nucleotide sequence of the gene is shown as SEQ ID NO.1. The gene has high toxicity on lepidoptera pests and is applied to transformation of microorganisms and plants, so that the microorganisms and the plants have toxicity on the related pests, and drug resistance of the pests on engineering bacteria and transgenic plants is overcome and delayed.

Description

technical field [0001] The invention relates to the technical field of biological control, in particular, the invention further relates to a Bt cry1Ca gene with high toxicity to lepidopteran pests and a protein encoded by the gene. Background technique [0002] Bacillus thuringiensis (Bt) is a widely distributed Gram-positive bacterium, an entomopathogenic microorganism with strong toxicity to pests and no toxicity to natural enemies, and no toxicity to higher animals and humans. It is currently the most in-depth study and the most widely used microbial insecticide, and it is active against more than 3000 kinds of pests in 16 orders. Bt can form insecticidal crystal proteins (Insecticidal Crystal Proteins, ICPs), also known as δ-endotoxin (delta-endotoxin) during the sporulation stage, its shape, structure and size are closely related to its virulence [Schnepf.E, Crickmore .N, Van Rie.J., Lereclus.D, Baum.J, Feitelson.J, Zeigler.D.R., Dean.D.H.Bacillus thuringiensis and its...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20A01N63/00A01P7/04C07K14/325C12N15/32C12N15/63C12N1/21A01N47/44C12R1/07
Inventor 高继国李海涛刘荣梅张杰宋福平
Owner NORTHEAST AGRICULTURAL UNIVERSITY
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com