Insecticide gene and application thereof

A technology of insecticidal genes and insecticidal proteins, which can be used in insecticides, genetic engineering, plant genetic improvement, etc., and can solve the problems of unsatisfactory expression and virulence.

Active Publication Date: 2012-07-04
BEIJING DABEINONG BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to provide a kind of insecticidal gene and its application, which is optimized and transformed according to the preferred codon of corn, and effectively overcomes the unsatisfactory expression and toxicity of the Cry1Ab insecticidal protein in corn in the prior art and other technical defects

Method used

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  • Insecticide gene and application thereof
  • Insecticide gene and application thereof
  • Insecticide gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

no. 1 example

[0048] The first embodiment, selection, optimization and synthesis of natural CrylAb gene sequence

[0049] 1. Select the natural Cry1Ab gene sequence for optimal transformation

[0050] The nucleotide sequence of the natural Cry1Ab8 gene with the sequence number AAA22551 registered by Oeda et al. in 1993 was selected as the basis for the optimization and transformation of the insecticidal gene of the present invention. The nucleotide sequence of the natural Cry1Ab8 gene is as SEQ ID NO: 2 in the sequence table As shown, the amino acid sequence of the natural Cry1Ab8 gene is shown in SEQ ID NO: 3 in the sequence listing.

[0051] 2. Intercept the natural Cry1Ab gene sequence for optimization and transformation

[0052] The nucleotide sequence corresponding to the 1st to 615th positions of the amino acid sequence encoded by the nucleotide sequence of the natural Cry1Ab8 gene is intercepted.

[0053] 3. Optimizing and transforming the above-mentioned truncated natural Cry1Ab g...

no. 2 example

[0059] The second embodiment, construction of recombinant expression vector and transformation of recombinant expression vector into Agrobacterium

[0060] 1. Construction of recombinant cloning vector DBN01Ab-T containing optimized Cry1Ab gene sequence

[0061]The synthetic optimized Cry1Ab gene sequence was connected into the cloning vector pGEM-T (Promega, Madison, USA, CAT: A3600), and the operation steps were carried out according to the instructions of the pGEM-T vector produced by Promega Company to obtain the recombinant cloning vector DBN01Ab-T, which was constructed Process such as figure 1 Shown (wherein, Amp represents the ampicillin resistance gene; f1 represents the replication origin of phage f1; LacZ is the LacZ initiation codon; SP6 is the SP6 RNA polymerase promoter; T7 is the T7 RNA polymerase promoter; mCry1Ab is the optimized Cry1Ab gene sequence (SEQ ID NO: 1); MCS is multiple cloning site).

[0062] Then, the recombinant cloning vector DBN01Ab-T was tr...

no. 3 example

[0072] The third embodiment, the acquisition and verification of corn plants transferred to the optimized Cry1Ab gene sequence

[0073] 1. Obtain corn plants transferred to the optimized Cry1Ab gene sequence

[0074] According to the routinely adopted Agrobacterium infection method, the immature embryos of the aseptically cultured corn variety Zong 31 (Z31) were co-cultured with the Agrobacterium described in 4 in the second embodiment, so that 2 and 3 in the second embodiment T-DNA (including the promoter sequence of corn Ubiquitin gene, optimized Cry1Ab gene sequence, known optimized Cry1Ab gene sequence, PMI gene and Nos terminator sequence) in the constructed recombinant expression vectors DBN100053 and DBN100053R (known sequence) was transfected Into the maize genome, obtained the maize plant transformed into the optimized Cry1Ab gene sequence and the maize plant transformed into the known optimized Cry1Ab gene sequence (positive control); at the same time, the wild type ...

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Abstract

The invention relates to insecticide gene and application thereof. A nucleotide sequence of the insecticide gene comprises a sequence (a) or a sequence (b), wherein the (a) has a nucleotide sequence shown in an SEQ ID NO: 1, and the (b) is a nucleotide sequence cross bred with the nucleotide sequence limited by the (a) on strict conditions. The insecticide gene is suitable for being expressed in corns, not only remarkably improves protein expression index and stability of CrylAb insecticide protein, and remarkably enhances toxicity of the CrylAb insecticide protein on main insects of corns.

Description

technical field [0001] The invention relates to an insecticidal gene and its application, in particular to a modified Cry1Ab insecticidal gene and its application. Background technique [0002] Plant pests are the main factor leading to the loss of crops, causing significant economic losses to farmers, and even affecting the living conditions of the local population. In order to control plant pests, people usually use broad-spectrum chemical insecticides and biological insecticides, but both have limitations in practical application: chemical insecticides will cause environmental pollution problems and lead to the emergence of resistant insects. However, biopesticides are easily degraded in the environment and require repeated application in production, which greatly increases production costs. [0003] In order to solve the limitations in the practical application of chemical insecticides and biological insecticides, scientists have discovered through research that by tran...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/32C12N15/63C12N5/10C12N1/21C12N1/19C12N7/01C12N15/82A01H5/00A01N47/44A01P7/04
Inventor 丁德荣庞洁康越景杨进孝贾志伟黄金存郭函子
Owner BEIJING DABEINONG BIOTECHNOLOGY CO LTD
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