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Artificially-synthesized Bt insecticidal protein gene Cry1Ab-GM1 and expression vector and application of gene

A technology of cry1ab-gm1 and insecticidal protein, applied in the field of genetic engineering, can solve the problems of silencing insect-resistant gene expression and low insect resistance of transgenic corn, so as to reduce usage, broad application space and market prospects, and reduce environmental pollution Effect

Active Publication Date: 2019-08-13
广州哈维种业有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The technical problem to be solved in the present invention is to provide a kind of synthetic Bt insecticidal protein gene Cry1Ab-GM1 , its expression vector, and applied to transgenic plants to solve the technical problems that insect resistance genes are easily expressed and silenced in corn, and transgenic corn has low insect resistance

Method used

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  • Artificially-synthesized Bt insecticidal protein gene Cry1Ab-GM1 and expression vector and application of gene
  • Artificially-synthesized Bt insecticidal protein gene Cry1Ab-GM1 and expression vector and application of gene
  • Artificially-synthesized Bt insecticidal protein gene Cry1Ab-GM1 and expression vector and application of gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Modification of insect resistance genes Cry1Ab gene

[0035] Based on his long-term extensive experiment and practical experience, the inventor Cry1Ab (GenBank sequence number is AY847289.1) Based on the insect-resistant gene, the codons are comprehensively upgraded and modified, and the codon-optimized insect-resistant gene is designed according to the coding characteristics of monocots. Cry1Ab-GM1 , Its nucleotide sequence is shown in SEQ ID NO.1.

[0036] Modified and synthesized insecticidal protein of Bacillus thuringiensis Cry1Ab-GM1 Gene, and original Cry1Ab Compared with the sequence of genes, it has the following remarkable features:

[0037] (1) Considering multiple factors, the modified gene is designed with specific deletions and DNA sequence changes, which greatly enhances its expression in plants;

[0038] (2) The use of plant-preferred codons reduces the original Cry1Ab Inverted repetitive sequences and AT-rich sequences in gene DNA sequences, and...

Embodiment 2

[0043] Example 2: Construction of recombinant expression vector and recombinant expression bacteria, the specific operation steps are as follows:

[0044] (1) Artificially modified synthetic insect resistance genes Cry1Ab-GM1 Genetic design Xba I. Sac I restriction site (5’ end plus Xba I restriction site, 3’ end Sac I restriction site), synthesized by Shenggong Bioengineering (Shanghai) Co., Ltd.;

[0045] (2) Use Xba I and Sac I double enzyme digestion synthesis Cry1Ab-GM1 Gene, and then recover gene fragments;

[0046] (3) The connection has been used Xba I, Sac I double-enzyme digestion treatment on the genetic transformation efficient plant expression vector pCAMBIA3300 to obtain Cry1Ab-GM1 The recombinant expression vector of the gene, the recombinant plasmid was named pCAMBIA3300-Cry1Ab-GM1;

[0047] Among them, the T-DNA of the pCAMBIA3300 vector is derived from the T-DNA region of the Ti plasmid pti37, but tumor-causing genes and sequences unrelated to T-DNA tra...

Embodiment 3

[0049] Example 3: Recombinant Agrobacterium-mediated transformation of maize immature embryos and callus

[0050] 1. Strip corn immature embryos

[0051] (1) Remove corn bracts;

[0052] (2) Cut the top of the fruit ear about 1cm, use the tweezers to insert the fruit ear from the top, use the tweezers as a handle, and then put the fruit ear into a beaker containing disinfectant. According to actual needs, you can put 4 to 6 in the same beaker Ears

[0053] (3) Add about 700mL of disinfectant (50% bleach or 5.25% sodium hypochlorite, and add a drop of Tween20) to the beaker to soak the ears and disinfect for 20 minutes; during the disinfection, rotate the ears from time to time while gently tapping the beaker to drive away the seeds Air bubbles on the surface to achieve the best disinfection effect;

[0054] (4) After disinfection, take out the ears and put them in a beaker filled with sterilized water, wash them in water 3 times, and then prepare to peel off the embryo;

[0055] (5) Pu...

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Abstract

The invention discloses an artificially-synthesized Bt insecticidal protein gene Cry1Ab-GM1 and an expression vector and application of the gene, and aims at solving the technical problems that an insect-resistant gene is subjected to loss of expression in corn, and transgenic corn is low in insect resistance. The artificially-synthesized Bt insecticidal protein gene Cry1Ab-GM1 is designed, the eukaryotic expression vector used for genetic transformation of plants is further designed, and the vector comprises an above gene sequence. An engineering bacterium used for genetic transformation of the plants is constructed and comprises the above gene sequence. The artificially-synthesized Bt insecticidal protein gene and the eukaryotic expression vector and the engineering bacterium thereof areapplied to transgenic plants. The artificially-synthesized Bt insecticidal protein gene Cry1Ab-GM1 is provided for the first time, can express in the corn and significantly provide the insect resistance for corn leaves and has wide application space and a wide market prospect in the aspect of providing the insect resistance of plants.

Description

Technical field [0001] The invention relates to the technical field of genetic engineering, in particular to an artificially synthesized Bt insecticidal protein gene Cry1Ab- GM1 , Its expression vector and its application. Background technique [0002] Bacillus thuringiensis ( Bacillus thuringiensis , Bt) belongs to the genus of prokaryotic bacteria, Bacillus, Bacillus, and is a Gram-positive bacteria. It was separated from the body fluid of the infected silkworm in 1901, and it was found to have a toxic effect on some lepidopteran insects. The insecticidal principle is: in the process of forming spores, a series of insecticidal protein crystals with highly specific toxic effects to insects are produced. Because the insecticidal protein crystals have no biological activity, they are called prototoxins and are sensitive. After insects ingest the prototoxin, under alkaline conditions in the midgut, the prototoxin is hydrolyzed into polypeptides. These polypeptides have specific ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/325C12N15/32C12N15/82A01H5/00A01H6/46
CPCC07K14/325C12N15/8286
Inventor 铁健雄
Owner 广州哈维种业有限公司
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