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Reforming composite insecticidal crystalline gene Cry2A of bacillus thuringiensis

A Bacillus thuringiensis gene technology, applied in the field of transformation and synthesis of Bacillus thuringiensis insecticidal crystal protein gene Cry2A, can solve the problems of incomplete transcripts, low protein expression, low Bt gene expression, etc.

Inactive Publication Date: 2006-03-08
HUAZHONG AGRI UNIV
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AI Technical Summary

Problems solved by technology

However, the insect resistance of these early Bt transgenic plants obtained by them is very weak, it is difficult to detect mRNA transcription, and the protein expression level is very low
There are many reasons for the low expression of Bt gene in plants: for example, 1, the wild Bt gene is rich in AT sequence, and the mRNA expressed in plants is unstable; 2, there may be eukaryotic gene inclusion in the wild Bt gene cleavage sites and transcription termination signal sequences, resulting in incomplete transcripts or abnormal processing of transcripts; 3. Microbes and plants have great differences in the frequency of codon usage in translation, which reduces translation efficiency; 4. True The 5'-UTR sequence of nuclear genes is very different from that of prokaryotic genes, and the 3' end of eukaryotic genes needs to be tailed to identify the signal sequence

Method used

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  • Reforming composite insecticidal crystalline gene Cry2A of bacillus thuringiensis
  • Reforming composite insecticidal crystalline gene Cry2A of bacillus thuringiensis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Plant gene and Cry2A codon bias analysis:

[0033] The nucleotide coding sequence of the original Cry2A gene was searched from Genbank, 984 plant gene coding sequences and 20 highly expressed ribosomal protein gene coding sequences were found, and the codon usage frequency of each type of gene was counted respectively, as shown in Table 1. It can be found that the Cry2A gene and the plant gene have a large difference in the use of codons, which is mainly manifested in: the Cry2A gene has a preference for the codon whose third wobble base is A or T, while the plant gene codon The third swing base prefers G or C.

[0034] Table 1 Statistics of codon usage frequency of various genes

[0035] Codon amino acid plant gene plant ribosomal protein gene original Cry2A

[0036] TAA $292 5 1

[0037] TGA $ 465 5 0

[0038] TAG $ 227 10 0

[0039] GCT A 7061 80 9

[0040] GCC A 10110 181 7

[0041] GCA A 5519 38 9

[0042] GCG A 7736 89 5

[0043] TGT C...

Embodiment 2

[0100] Example 2: Codon modification of Cry2A gene and codon feature analysis of the new Cry2A gene synthesized

[0101] According to the analysis results in Table 1, the corresponding codons of the original Cry2A were replaced with the preferred codons of the plant genes, the AT-rich sequences and ambiguous intron sequences such as ATTTA and AATGAA in the Cry2A gene were eliminated, and the large intron sequences in the gene were excluded. The inverted repeat sequence and common restriction endonuclease recognition site sequence; the coding sequence of the designed and synthesized Cry2A gene is shown in the sequence table SEQ ID NO..1. The codon characteristics of the target synthesized Cry2A gene are shown in Figure 2.

Embodiment 3

[0102] Example 3: Analysis of the characteristics of the new Cry2A coding sequence synthesized

[0103] The original Cry2A and the synthetic new Cry2A coding sequence were analyzed by Blast2, and the homology of the two sequences was 69.45%. The statistical results of the base composition are: the C+G% of the original gene is 34.75%, and the C+G% of the newly synthesized gene is 59.04%. The Blast2 analysis of the encoded amino acid sequences showed that the encoded amino acid sequences of the two were completely consistent. Embodiment 4 Improves the addition of the terminal sequence of the stability and expression efficiency of gene transcripts in plant cells

[0104] Sequence 2 was designed by analyzing the structure of the 5' guide sequence of the plant gene, which is shown in the sequence listing SEQ ID NO..2, and added to the 5' end of the coding sequence of the new Cry2A gene. Another designed sequence is shown in the sequence table SEQ ID NO..3, which is added at the 3...

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Abstract

A Bacillus thuringiensis insecticidal crystal protein DNA sequence Cry2A is designed and synthesized. Comparing it with original Cry2A DNA sequence can show that the amino acid components of DNa sequence coding protein are not changed, the application frequency of vegetable preference codon is higher and the AT sequence, reverse reduplicate sequence and indefinable subsequences are eliminated. It can be used for culturing the seed of insect-resisting transgenic plant.

Description

technical field [0001] The invention belongs to artificially transforming and synthesizing the DNA sequence of the Bacillus thuringiensis (Bt) insecticidal crystal protein (ICP) gene and a preparation method thereof. The DNA sequence of the present invention can be used as the technical field of molecular breeding for high-efficiency expression in transgenic insect-resistant plants. Background technique [0002] Insect infestation is an important factor causing loss of agricultural production. According to statistics, the direct economic loss caused by insect pests to agricultural production is as high as 13% every year. [0003] Chemical pesticides have made important contributions to pest control and stable agricultural production. With people's awareness of the environmental hazards of chemical pesticides and the increasing awareness of environmental protection, environmentally safe biological pesticides have become a research hotspot. Amo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/32C12N1/21C12N15/29C12N15/63A01N63/00
Inventor 林拥军张启发
Owner HUAZHONG AGRI UNIV
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