Pesticidal protein encoding gene Cry1Ab-Ma and expression vector and application thereof

A cry1ab-ma, insect-resistant gene technology, applied in the fields of application, genetic engineering, plant genetic improvement, etc., can solve the problems of ecological imbalance, lack of insect-resistant varieties, environmental pollution, etc., to reduce the amount of use, enhance expression, reduce Effects of Environmental Pollution

Inactive Publication Date: 2011-06-15
INST OF CROP SCI CHINESE ACAD OF AGRI SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the lack of suitable insect-resistant varieties, the current main method to solve insect pests is to spray chemical insecticides during the growth process; however, chemical insecticides kill pests and their natural enemies at the same time, causing ecological imbalance and environmental pollution

Method used

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  • Pesticidal protein encoding gene Cry1Ab-Ma and expression vector and application thereof
  • Pesticidal protein encoding gene Cry1Ab-Ma and expression vector and application thereof
  • Pesticidal protein encoding gene Cry1Ab-Ma and expression vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Synthesis of embodiment 1Cry1Ab-Ma gene

[0031] According to the original nucleotide sequence of the Bt gene Cry1Ab, a base sequence of 1623bp at the 3' end was removed, leaving only a partially deleted base sequence of 1845bp at the 5' end; In the case of changes, base substitutions are carried out with plant-favored codons, and a modified DNA sequence is initially obtained; AT-rich sequences (such as ATTTA, AATGAA, etc.) and common restrictions that exist in the DNA sequence that cause plant transcription instability are excluded SacI, and then correct and eliminate it by replacing the codon; and add a stop codon TAG at the 3' end; determine and chemically synthesize the transformed Bt gene, its nucleotide sequence is as shown in SEQ ID No .1 shown; according to the needs of cloning, add restriction endonuclease recognition site sequences at both ends of the sequence and construct it into the corresponding expression vector.

[0032] The original Bt gene Cry1Ab was co...

Embodiment 2

[0034] Example 2 Expression of Cry1Ab-Ma gene in prokaryotic system and detection of product toxicity

[0035] In order to detect the in vitro expression of the modified Cry1Ab-Ma gene and its toxicity to the corn borer, we constructed a Bt prokaryotic expression vector. According to the needs of cloning the Bt gene, the NdeI endonuclease recognition site sequence CATATG was added to the 5' end of the primer sequence, and the HindIII endonuclease recognition site sequence AAGCTT was added to the 3' end. Primer sequences were designed (upstream primer F1: 5"-CATATGGACAACAACCCGAACATC-3'; downstream primer R1: 5'-AAGCTTCTAGTACTCCGCCTCG-3').

[0036] Using the pUCAb-Ma plasmid as a template, using F1 and R1 as primers, the Cry1Ab-Ma gene was amplified, digested with restriction endonucleases NdeI and HindIII, and the gel recovery kit recovered and purified the Cry1Ab-Ma gene fragment. Digest pET28b+ with restriction endonucleases NdeI and HindIII, and recover and purify the 5.3kb...

Embodiment 3

[0048] Example 3 Expression of Cry1Ab-Ma gene in transgenic plants and detection of toxicity of expression products

[0049] The T vector plasmid pUCCry1Ab-Ma was digested with XbaI and SacI, and the Cry1Ab-Ma fragment was recovered with a gel extraction and purification kit. At the same time, the plant expression vector CPB was double-digested with XbaI and SacI, and the digested CPB fragment was recovered with a gel extraction and purification kit. The two fragments were ligated to construct the eukaryotic expression plasmid pCAMBIA1300-Cry1Ab-Ma-Bar (the promoter is CaMV35S, the marker gene is the herbicide resistance gene Bar of Streptomyces hygroscopicus). Restriction identification showed that the vector was constructed correctly.

[0050] Among them, CPB (pCAMBIA1300-35S-MCS-Bar, figure 2 ) plasmid is a plasmid constructed on the basis of pCAMBIA1300 plasmid, which contains a target gene insertion cassette (35S-multicloning restriction site-Tnos) and a herbicide resi...

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Abstract

The invention provides an insect-resistant gene Cry1Ab-Material. The synthesis of the gene is as follows: according to the amino acid sequence of the N-terminal of the original Bacillus thuringiensis (Bt) protein (Cry1Ab), the preferred codons of monocotyledon (corn) is used to perform human reformation and synthetize a new Cry1Ab DNA sequence; and the prokaryotic expression vector and plant expression vector are constructed, and transformation is performed in the host cells. Vitro tests prove that the transformed and synthetized Bt gene toxic protein has obvious insecticidal effect on Ostrinia nubilalis. The expression of the insect-resistant gene Cry1Ab-Ma in monocotyledon is stable and efficient, thus the gene can be used to produce insect-resistant transgenic plant.

Description

technical field [0001] The invention relates to the field of genetic engineering and biological control, in particular to insect-resistant gene Cry1Ab-Ma, its expression vector and application. Background technique [0002] The Bt gene encodes an insecticidal crystal protein from Bacillus thuringiensis (Bacillus thuring Hansis). It produces delta-endotoxin insecticidal parasporal crystal proteins during sporulation, and these proteins have high insecticidal activity. Its principle of action is that this insect-resistant protein can be dissolved by alkaline intestinal fluid and hydrolyzed into smaller active toxin fragments - core fragments (Hofte and Whiteley, 1989). The active fragment is resistant to further hydrolysis by proteases, and the activated protein binds to the brush vesicles in the insect gut, causing perforation and affecting osmotic balance, cells swell and dissolve, target organisms stop feeding and eventually die. Studies have shown that the intestinal epi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/32C07K14/325C12N15/82
CPCY02A40/146
Inventor 李新海铁双贵岳润清翁建峰谢传晓郝转芳
Owner INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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