Insect-resistant fusion gene mCry1AbVip3A, and expression vector and application thereof

A technology of fusion gene and gene, which is applied in the field of genetic engineering and biological control, can solve the problems that fusion protein fusion protein cannot be folded correctly, does not show synergistic effect, and only maintains insecticidal activity, etc., and achieves reduced usage and important economic value , the effect of reducing environmental pollution

Inactive Publication Date: 2020-07-24
INST OF CROP SCI CHINESE ACAD OF AGRI SCI +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Saraswathy et al. constructed a chimeric protein of Vip3Aa14 and Cry1Ac. The chimeric protein mainly exists in the form of inclusion bodies and has good stability to trypsin, but only maintains the insecticidal activity of Cry1Ac and does not show a synergistic effect.
In addition, the C-terminus of Cry1C can significantly enhance the synthesis of Vip3Aa7 and help it form inclusion bodies in BMB171, but its biological activity is significantly lower than that of protoxin Vip3Aa7 (Song R et al., 2008)
The decreased toxicity of the above fusion protein may be caused by the inability of the fusion protein to fold correctly

Method used

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  • Insect-resistant fusion gene mCry1AbVip3A, and expression vector and application thereof
  • Insect-resistant fusion gene mCry1AbVip3A, and expression vector and application thereof
  • Insect-resistant fusion gene mCry1AbVip3A, and expression vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Synthesis of embodiment 1 insect-resistant fusion gene

[0029] According to the original nucleotide sequence of the Vip3A gene (gene ID DQ539888.1), while keeping the amino acid composition of its protein unchanged, base substitutions were performed with codons preferred by monocotyledonous plants, and a modified DNA sequence was preliminarily obtained ; Get rid of the AT sequence (such as ATTTA, AATGAA etc.) and the commonly used restriction enzyme cutting site (SacI) that cause plant transcription instability to exist in the DNA sequence, then correct and eliminate by replacing the codon; and in 5 Add the Cry1Ab-Ma sequence at the 'end; determine and chemically synthesize the modified insect-resistant fusion gene mCry1AbVip3A, whose nucleotide sequence is shown in SEQID No.1; add restriction endonuclease recognition sites at both ends of the sequence according to the needs of cloning point sequence and constructed to the corresponding expression vector.

Embodiment 2

[0030] Example 2 Expression of insect-resistant fusion gene in prokaryotic system and detection of toxicity of product

[0031] In order to detect the in vitro expression of the modified insect-resistant gene and its toxicity to corn borer, we constructed the prokaryotic expression vector pET-mCry1AbVip3A.

[0032]Design primer sequence (upstream primer F1: as shown in SEQ ID No.3, 5'-GACGACGACAAGGCCATGGATGGACAACAACCCG-3'; F2: as shown in SEQ ID No.4, 5'-ACGACGACAAGGCCATGGATGAACAAGAACAAC-3'; downstream primer R1: 5' -GGTGGTGGTGGTGCTCGAGCTTGATGCTCACGTC-3', as shown in SEQ ID No. 5; R2: as shown in SEQ ID No. 6, 5'-GGTGGTGGTGGTGCTCGAGGTACTCCGCCTCGAA-3').

[0033] The constructed pTF101.1-mCry1AbVip3A plant expression plasmid ( figure 1 ) as a template, using F1 and R1 as primers to amplify the insect-resistant fusion gene, and the gel recovery kit (Tiangen Biochemical Technology (Beijing) Co., Ltd., DP209) recovered and purified the above insect-resistant gene fragments. Restr...

Embodiment 3

[0049] Example 3 Expression of mCry1AbVip3A gene in transgenic maize

[0050] pTF101.1-mCry1AbVip3A ( figure 1 ) insertion sequence is introduced into the callus of recipient maize, and the transgenic maize is obtained after being screened by the herbicide bialaphos. Detection of target protein mCry1AbVip3A (Bt-Cry1Ab / 1Ac colloidal gold immunological detection):

[0051] ①Take about 1cm 2 The left and right fresh young leaves were placed in a 1.5ml Eppendorf tube, and 500ul of SEB4 sample extraction buffer was added to the tube and ground.

[0052] ②Take out the test strip (Beijing Zenith Biotechnology Co., Ltd., article number: STX06200 / 0050), hold the top of the test strip, and insert the marked end into the centrifuge tube. The quality control line will appear within 3-5 minutes, and if the sample is positive, the test line will appear.

[0053] The expression of mCry1AbVip3A in the leaves of 6 transgenic plants was detected by colloidal gold test strip detection method...

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Abstract

The invention discloses an insect-resistant fusion gene mCry1AbVip3A. The nucleotide sequence of the gene mCry1AbVip3A is as follows: (a) nucleotide as shown in SEQ ID NO:1; or (b) a nucleotide encoding an amino acid represented by SEQ ID NO:2; or (c) a nucleotide which can be hybridized with a complementary sequence of SEQ ID NO:1 under strict hybridization conditions, wherein the protein coded by the nucleotide has a function of an mCry1AbVip3A transcription factor. In-vitro toxicity determination tests prove that the modified and synthesized fusion gene toxin-producing protein has a remarkable insecticidal effect on ostrinia nubilalis and spodoptera frugiperda, can be stably and efficiently expressed in monocotyledons, and is further used for producing insect-resistant transgenic plants.

Description

technical field [0001] The present invention relates to the fields of genetic engineering and biological control, in particular, it relates to an insect-resistant fusion gene, its expression vector and its application Background technique [0002] Pests cost global agricultural production an estimated $8 billion per year. The control of pests currently mainly relies on the use of chemical pesticides, but pesticide residues will cause harm to human health. Therefore, people have successfully chosen to use transgenic crops that can produce insecticidal toxins for insect resistance. At present, transgenic insect-resistant corn and cotton have been planted in large quantities. [0003] Substances with strong insecticidal activity against pests such as Agrotis ipsilon, Spodoptera exigua and Spodoptera frugiperda were found in the fermentation supernatant of strain Bt AB88. It is expressed from the mid-growth stage to the budding stage, and it is named as vegetative insecticidal...

Claims

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Application Information

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IPC IPC(8): C12N15/62C12N15/82C12N1/21C12N5/10A01H5/00A01H6/46A01H6/82A01H6/60C12R1/01
CPCC07K14/325C07K2319/00C12N15/8286C12N2800/22C12N2800/80
Inventor 翁建峰李新海宋新元武奉慈李望舒
Owner INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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