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

Optimization of pest resistance genes using DNA shuffling

A technology for resistance genes and pests, applied in recombinant DNA technology, genetic engineering, organic chemistry, etc., can solve problems such as lack of activity

Inactive Publication Date: 2001-09-26
MAXYGEN
View PDF45 Cites 15 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the greatest limitation of many of these genes used is lack of sufficient activity (potency) and / or lack of a useful spectrum of activity

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
  • Optimization of pest resistance genes using DNA shuffling
  • Optimization of pest resistance genes using DNA shuffling
  • Optimization of pest resistance genes using DNA shuffling

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0268] Example 1: Optimization of CRYl toxin with DNA shuffling

[0269] The cry1C gene including its own promoter (5' region to -260 nucleotides) was used as a substrate for DNA shuffling. After DNA shuffling, the protein coding region was cloned into an expression vector and transformed into E. coli cells. The transformed cells were incubated in a bacterial culture medium (nutrient broth) at 30°C for 72 hours, and then the cells formed inclusion bodies composed of Cry1C protein. Then the cells are harvested by centrifugation or filtration, lysozyme is used to lyse, and free inclusion bodies are released. Alternatively, lysis can be achieved by detergent treatment, sonication, or other methods known to those skilled in the art. The inclusion bodies are collected by centrifugation or filtration, and then exposed to an alkaline solution (pH 10.5) with or without a disulfide bond reducing agent (such as 2-mercaptoethanol). Then trypsin is used to activate the Cry 1C protein dissolve...

Embodiment 2

[0272] Example 2: Reorganization of the insecticidal toxin gene of Bacillus japonicus

[0273]Bacillus japonicus is known to be a pathogen of scarab beetles (such as Japanese beetles), which produces an insecticidal protein called Cry18Aa (Zhang et al. (1997) J. Bacteriol. 179: 4336-4341). However, the insecticidal activity of the protein is not high enough to be used on a large scale to prevent crop damage caused by beetle infestation. This example describes the optimization of Cry18Aa by shuffling the cry18Aa gene of Bacillus japonicus and its Bacillus thuringiensis homologous gene cry2.

[0274] The cry18Aa gene was amplified by polymerase chain reaction (PCR) from Bacillus japonicus with two primers. The two primers were designed according to the published sequence (GenBank accession number: X99049). The forward primer (5'-gaaggaggctattggCCatgGac-3') is based on the ribosome binding site and the sequence surrounding the translation initiation signal. The sequence was modified ...

Embodiment 3

[0280] Example 3: Cloning of previously unknown genes from insect pathogens encoding insecticidal proteins

[0281] Genomic DNA is prepared from several insect pathogens, such as Pseudomonas aeruginosa and Serratia entomophilus. The DNA samples were digested with several enzymes including NotI, BamHI and SphI. The fragments produced by these enzymes are graded according to size, and cloned into a cosmid vector (such as Supercos (Stratagene)) or lambda vector (such as λZAP (Stratagene)) according to size. The tomato hornworm and cucumber beetle were then used to screen the E. coli library containing insect pathogen DNA for insecticidal activity. E. coli cells were cultured in LB broth at 30°C for 48 hours and harvested by centrifugation. The pelleted cells are resuspended in a minimum amount of water and placed on insect food. Allow insects to eat the food for 3 days. Several cosmid clones showing insecticidal activity were identified, and the DNA was isolated.

[0282] The cosmid ...

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 present invention provides methods for obtaining pest resistance genes that are improved over naturally occurring genes for conferring pest resistance to plants. The method involves the use of DNA shuffling of pest resistance genes to generate a library of recombinant pest resistance genes, and then screening the library to identify those genes that show an improved property of interest.

Description

[0001] Cross-documentation of related applications [0002] This application claims priority of 09 / 071,816 filed on May 1, 1998 (transformed into provisional application No. 60 / 122,054) and provisional application 60 / 094,462 filed on July 28, 1998. Invention field [0003] The present invention relates to the field of developing optimized genes that can confer resistance to insects, nematodes, fungi and other pests in plants. Background of the invention [0004] Genes encoding proteins with insecticidal activity are currently used for agricultural control of specific pests (AsgrowReports-"Genetic Engineering for Controlling Pests"-Len Copping, Chapter 2.1-2.4). For example, the gene encoding Bacillus thuringiensis (Bt) crystal protein has been stably incorporated into several crops and is widely used as an insect control agent (Pest. Sci. (1998) 52:165-175, Asgrow Reports, supra) . Several other differen...

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
IPC IPC(8): C12N15/09C07K14/325C12N5/00C12N15/82C12R1/07
CPCC07K2319/00C12N15/8286C07K14/325C12N15/8285Y02A40/146C12N15/8279
Inventor W·P·施特默尔L·卡斯尔山本高石
Owner MAXYGEN
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