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Artificial synthetic high gene order expression high virulence protein for lepidoptera pest and use thereof

A technology of gene sequence and artificial synthesis, applied in the fields of application, plant gene improvement, and microbial-based methods, can solve problems such as reduced protein translation efficiency, abnormal mRNA shearing, and unstable expression products

Inactive Publication Date: 2009-02-04
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in transgenic research, it has been found that the insecticidal protein gene derived from Bt is directly transferred into plants, and there are defects of unstable expression products and low expression levels (vanAarssen, et al, 1995, Plant Mol Biol, 28: 513-524 )
Specific problems include: 1) the natural Bt gene contains high AT, more than 60%, and the mRNA expressed by such a gene in the plant is very easy to be degraded by the plant; 2) there are intron cut points similar to eukaryotic genes in the natural Bt gene, Transcription terminator sequence, resulting in incomplete transcription, abnormal splicing of mRNA, etc.; 3) The codons used in natural Bt genes are quite different from those in plants, which will reduce the efficiency of protein translation; 4) Natural Bt genes are used as prokaryotic sources The structure of the gene is significantly different from that of plants and other eukaryotes, such as eukaryotes containing 5'-UTR sequence and polyA tail sequence at the 3' end

Method used

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  • Artificial synthetic high gene order expression high virulence protein for lepidoptera pest and use thereof
  • Artificial synthetic high gene order expression high virulence protein for lepidoptera pest and use thereof
  • Artificial synthetic high gene order expression high virulence protein for lepidoptera pest and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] Example 1 Transformation and synthesis of the crylAh gene for common plant transformation

[0081] According to the amino acid sequence (SEQ NO.1) of the crylAh gene (Chinese patent, patent number: 200410009918.9), the present invention first adopts the 1-2001bp sequence (SEQ NO. NO.2) Carry out artificial optimization and transformation. Try to avoid using rare plant codons, and adjust the codon usage frequency (Table 1), so that the codon usage frequency of CrylAh protein is close to the usage frequency in plants (Table 1). On this basis, the typical AT-rich sequences that cause instability of plant gene transcripts in the DNA sequence were removed, and the hairpin structure and common restriction enzyme sites were removed, in order to form a In NcoI (CCATGG), an amino acid (glycine, GGA) was added after the first start codon, and the nucleotide sequence obtained was SEQ NO.3. The homology between this sequence and the crylAh gene is only 86.18%, and the G+C content...

Embodiment 2

[0086] Embodiment 2, transformation and synthesis of the crylAh gene for rice transformation

[0087] According to the codon preference of rice genes, the invention optimizes the codons for plants on the mcrylAh gene. According to the amino acid sequence (SEQ NO.3) of the mcrylAh gene, under the premise of ensuring that the amino acid sequence remains unchanged, artificially optimize and transform the SEQ NO.3 by adopting rice preferred codons. The frequency of codon usage was adjusted to make the frequency of codon usage of CrylAh protein close to that of rice genes. Finally, it was determined that the nucleotide sequence of the further modified mcrylAh gene, that is, the mrcrylAh gene, was shown in SEQ ID NO.7. For the convenience of subsequent gene manipulation, BamHI sites were added on both sides of the artificially synthesized gene. Finally, the nucleotide sequence of the further modified mcrylAh gene, namely the mrcrylAh gene, was determined, as shown in SEQ NO.5. The...

Embodiment 3

[0092] Expression of embodiment 3, mcrylAh gene and mrcrylAh gene in escherichia coli

[0093] Using the pTeasy-mcrylAh plasmid as a template, amplify the 2Kb PCR product with specific primers, recover and purify, digest with BamHI and Sal I, and pET-21b plasmid (commonly used plasmid, which can induce expression in Escherichia coli) with the same treatment Source gene, which can be purchased at Novagen Company) was connected, transformed into E.coli JM110, and identified by restriction analysis and PCR ( Figure 7 ), screen out positive recombinants, and name the obtained recombinant expression vector plasmid pET-1Ahp.

[0094] The recombinant plasmid pET-1Ahp was transformed into Escherichia coli BL21, 150rpm, 18°C ​​to induce the expression of the target protein, sonicated, centrifuged, and the supernatant and precipitate were respectively taken for SDS-PAGE (8%) analysis ( Figure 8 ). The mcrylAh gene can be expressed in E. coli.

[0095] The same procedure was used to...

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Abstract

The present invention relates to a gene sequence of an artificially synthesized protein expressing high toxicity to Lepidoptera pests and an application thereof in the field of plant protection, which belongs to the technical field of biological control. The gene sequence of the artificially synthesized protein expressing high toxicity to Lepidoptera pests is characterized in that a coding area is provided with a nucleotide sequence which is 81 percent similar to crylAh gene and can encode the same amino acid sequence as the crylAh gene does. The present invention adjusts the nucleotide composition of the crylAh gene, so that the crylAh gene can approach the codon usage frequency of plant genes but does not change the encoded amino acid sequence; by building the gene sequence obtained by the present invention into an appropriate backbone vector and converting a receptor plant, the gene sequence can be expressed in the plant; the result of a test shows that because of expressing the gene, a positive plant acquires resistibility to Lepidoptera pests.

Description

technical field [0001] The invention belongs to the technical field of biological control, in particular to an artificially synthesized gene sequence for expressing highly toxic proteins to lepidopteran pests and its application. Background technique [0002] Insect pests are an important factor in the reduction of crop production in the world. On average, 10% of the total grain output is lost every year, and the direct economic loss amounts to billions of dollars. The planting areas of rice, corn and cotton in my country are 28.37 million, 25.44 million and 5.69 million hectares respectively (2006, China Agricultural Information Network). The main pests are rice borer, corn borer and cotton bollworm, which seriously threaten these important food crops. and safe production of cash crops. In the past few decades, the control has mainly relied on chemical pesticides. While making great contributions to agricultural production, chemical pesticides have caused serious consequenc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/82A01H1/00A01H5/00C12N1/15C12N1/19C12N1/21C07K14/00C12R1/01C12R1/645
Inventor 张杰郎志宏宋福平何康来梁革梅黄大昉束长龙
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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