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Synthetic Bt insecticidal gene mcry1Ab for transgenosis insect-resistant plants

A technology of transgenic plants and transgenic cell lines, applied in the field of biological control, can solve problems such as lack of commercialization

Active Publication Date: 2017-11-24
北京粮元生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although there are some reports on the cultivation of transgenic insect-resistant corn varieties in my country, they have not been able to be commercialized, mainly due to the insect-resistant effect of the Bt gene and the stability of transformed plants.

Method used

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  • Synthetic Bt insecticidal gene mcry1Ab for transgenosis insect-resistant plants

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Embodiment 1, transformation of Cry1Ab gene and acquisition of mCry1Ab gene

[0079] The amino acid sequence of the Cry1Ab protein is shown in sequence 4 in the sequence listing. The nucleotide sequence of the gene encoding the Cry1Ab protein (hereinafter referred to as the Cry1Ab gene) is shown in sequence 3 in the sequence listing.

[0080] The inventors of the present invention carefully analyzed the active region of the Cry1Ab protein, and under the premise of ensuring further improvement of its expression level, modified the gene coding frame and codons of the Cry1Ab protein according to the coding characteristics of monocotyledonous plants. The modified Cry1Ab protein is named mcry1Ab protein. The amino acid sequence of the mcry1Ab protein is shown in sequence 2 in the sequence listing. The nucleotide sequence of the gene encoding the mcry1Ab protein (ie, the modified Cry1Ab gene, hereinafter referred to as the mcry1Ab gene) is shown in sequence 1 in the sequenc...

Embodiment 2

[0085] Example 2, bioassay of mcry1Ab protein to Lepidoptera insects

[0086] 1. In vitro expression and purification of mcry1Ab protein

[0087] The in vitro expression and purification steps of mcry1Ab protein are as follows:

[0088] 1. Artificially synthesize the double-stranded DNA molecule shown in sequence 1 in the sequence listing.

[0089] 2. Ligate the double-stranded DNA molecule synthesized in step 1 with the prokaryotic expression vector pEASY-E1 to obtain the recombinant plasmid pEASY-mcry1Ab.

[0090] The recombinant plasmid pEASY-mcry1Ab was sequenced. Sequencing results show that the recombinant plasmid pEASY-mcry1Ab contains the DNA molecule shown in sequence 1 in the sequence listing, and expresses the mcry1Ab protein shown in sequence 2 in the sequence listing.

[0091] 3. The recombinant plasmid pEASY-mcry1Ab was introduced into Escherichia coli transetta to obtain a recombinant bacterium, which was named transetta-mcry1Ab.

[0092] 4. Take a single cl...

Embodiment 3

[0103] Example 3. Obtaining and identification of insect resistance of transgenic mCry1Ab maize

[0104] 1. Construction of recombinant vector

[0105] The recombinant plasmid pCAMBIA3301+mCry1Ab was constructed and sequenced. According to the sequencing results, the structure of the recombinant plasmid pCAMBIA3301+mCry1Ab 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 table from the 1st to the 2458th position from the 5' end Double-stranded DNA molecules are shown. The recombinant plasmid pCAMBIA3301+mCry1Ab expresses the mcry1Ab protein shown in Sequence 2 in the Sequence Listing.

[0106] The expression cassette is contained in the recombinant plasmid pCAMBIA3301+mCry1Ab, 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 5...

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Abstract

The invention discloses a synthetic Bt insecticidal gene mcry1Ab for transgenosis insect-resistant plants. The Bt insecticidal gene mcry1Ab for transgenosis insect-resistant plants provided by the invention is used for encoding mCry1Ab protein. The mCry1Ab protein is b1), b2) or b3) as follows: b1) the protein with the amino acid sequence as shown in the sequence 2 in a sequence table; b2) the fused protein acquired in the manner of connecting a label with a N end or / and C end of the protein as shown in the sequence 2 in the sequence table; and b3) the protein related to plant insect resistance that is acquired in the manner of substituting and / or deleting and / or adding one or more amino acid residue of the amino acid sequence shown as the sequence 2 in the sequence table. An experiment proves that the mcry1Ab gene can easily acquire the transformant with high expression quantity than the cry1Ab gene, the cry1Ab protein, compared with the mcry1Ab protein, has obvious higher insecticidal activity to ostrinia nubilalis, oriental armyworm, cotton bollworm and dichocrocis punctiferalis, and the mcry1Ab gene has an important popularizing value.

Description

technical field [0001] The invention belongs to the technical field of biological control, and in particular relates to the Bt mcry1Ab 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 delta-endotoxin from Bacillus thuringiensis. In 1985, Adang, M.J et al. cloned the Cry1Ac gene from Bacillus thuringiensis, 1986 Wabiko, H. et al. cloned the cry1Ab gene from Bacillus thuringiensis, which encodes 1155 amino acids and ...

Claims

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Application Information

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IPC IPC(8): C07K14/32C12N15/32C12N15/82A01H5/00
CPCC07K14/325C12N15/8286
Inventor 赖锦盛赵海铭宋伟彬
Owner 北京粮元生物科技有限公司
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