Artificially-synthetic Bt insecticidal gene mcrylF for transgenic insect-resistant plant

A technology of transgenic plants and transgenic cell lines, applied in the field of biological control, can solve the problem of no commercialization

Active Publication Date: 2017-12-15
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although there are some reports on the cultivation of transgenic insect-resistant corn varieties in my country, they have not b

Method used

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  • Artificially-synthetic Bt insecticidal gene mcrylF for transgenic insect-resistant plant
  • Artificially-synthetic Bt insecticidal gene mcrylF for transgenic insect-resistant plant
  • Artificially-synthetic Bt insecticidal gene mcrylF for transgenic insect-resistant plant

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Experimental program
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Effect test

Embodiment 1

[0079] Embodiment 1, transformation of Cry1F gene and acquisition of mcry1F gene

[0080] The amino acid sequence of Cry1F protein is shown as sequence 4 in the sequence listing. The nucleotide sequence of the gene encoding Cry1F protein (hereinafter referred to as Cry1F gene for short) is shown as sequence 3 in the sequence listing.

[0081] The inventors of the present invention carefully analyzed the active region of the Cry1F protein, and under the premise of ensuring further improvement of its expression level, modified the gene coding frame and codons of the Cry1F protein according to the coding characteristics of monocotyledonous plants. The modified Cry1F protein was named mcry1F protein. The amino acid sequence of mcry1F protein is shown as sequence 2 in the sequence listing. The nucleotide sequence of the gene encoding mcry1F protein (that is, the modified Cry1F gene, hereinafter referred to as mcry1F gene) is shown in sequence 1 in the sequence listing.

[0082] ...

Embodiment 2

[0086] Example 2, bioassay of mcry1F protein on Lepidoptera insects

[0087] 1. In vitro expression and purification of mcry1F protein

[0088] The in vitro expression and purification steps of mcry1F protein are as follows:

[0089] 1. Artificially synthesize the double-stranded DNA molecule shown in sequence 1 in the sequence listing.

[0090] 2. Ligate the double-stranded DNA molecule synthesized in step 1 with the prokaryotic expression vector pEASY-E1 to obtain the recombinant plasmid pEASY-mcry1F.

[0091] The recombinant plasmid pEASY-mcry1F was sequenced. Sequencing results show that the recombinant plasmid pEASY-mcry1F contains the DNA molecule shown in sequence 1 in the sequence listing and expresses the mcry1F protein shown in sequence 2 in the sequence listing.

[0092] 3. The recombinant plasmid pEASY-mcry1F was introduced into Escherichia coli transetta to obtain a recombinant bacterium, which was named transetta-mcry1F.

[0093] 4. Take a single clone of tra...

Embodiment 3

[0104] Example 3. Obtaining and identification of insect resistance of transgenic mcry1F maize

[0105] 1. Construction of recombinant vector

[0106] The recombinant plasmid pCAMBIA3301+mcry1F was constructed and sequenced. According to the sequencing results, the structure of the recombinant plasmid pCAMBIA3301+mcry1F is described as follows: replace the small fragment between the recognition sequences of the restriction endonucleases HindIII and BstEII of the vector pCAMBIA3301 with the sequence 5 in the sequence listing from 1 to 2392 from the 5' end Double-stranded DNA molecules are shown. The recombinant plasmid pCAMBIA3301+mcry1F expresses the mcry1F protein shown in Sequence 2 in the Sequence Listing.

[0107] The expression cassette is contained in the recombinant plasmid pCAMBIA3301+mcry1F, and the nucleotide sequence of the expression cassette is shown in sequence 5 in the sequence list. In sequence 5 in the sequence listing, the 1st to 583rd from the 5' end is t...

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Abstract

The invention discloses an artificially-synthetic Bt insecticidal gene mcrylF for a transgenic insect-resistant plant. The artificially-synthetic Bt insecticidal gene mcrylF for the transgenic insect-resistant plant, disclosed by the invention, encodes mCry1F protein. The mCry1F protein is b1) or b2) or b3), wherein the b1) is protein with an amino acid sequence shown as a sequence 2 in a sequence table; the b2) is fusion protein obtained by connecting labels at the N end or/and C end of the protein shown as the sequence 2 in the sequence table; the b3) is plant insect resistance-related protein obtained by performing the replacement and/or the loss and/or the addition of one or several amino acid residues on the amino acid sequence shown as the sequence 2 in the sequence table. An experiment shows that compared with a crylF gene, the gene mcrylF more easily obtains a highly-expressed transformant; compared with those of crylF protein, the insecticidal activities of the mcrylF protein for ostrinia nubilalis, mythimna separate, cotton bollworms and dichocrocis punctiferalis are significantly improved; therefore, the popularization value is important.

Description

technical field [0001] The invention belongs to the technical field of biological control, and in particular relates to the Bt mcry1F gene used for plant expression. Background technique [0002] The Bt gene encodes an insecticidal crystal protein, which is derived from Bacillus thuringiensis, which is a Gram-positive Agrobacterium belonging to the genus Bacillus. It produces insecticidal parasporal crystal proteins called delta-endotoxins during sporulation (the gene that controls the synthesis of this protein is on a plasmid), and these proteins are harmful to Lepidoptera, Diptera, Coleoptera, Hymenoptera and other insects have specific insecticidal activity. [0003] In 1981, Schenpf and Whiteley cloned the first cry gene Cry1Aa1 encoding δ-endotoxin from Bacillus thuringiensis. In 1985, Adang, M.J et al. from Bacillus thuringiensis [0004] (Bacillus thuringiensis) cloned the Cry1Ac gene. In 1986, Wabiko, H. et al. cloned the cry1Ab gene from Bacillus thuringiensis. Th...

Claims

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Application Information

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IPC IPC(8): C07K14/325C12N15/32C12N15/82A01H5/00
CPCC07K14/325C12N15/8286
Inventor 赖锦盛赵海铭宋伟彬
Owner CHINA AGRI UNIV
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