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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, difficulty in detecting mRNA, mRNA instability, etc.

Inactive Publication Date: 2004-03-10
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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
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Effect test

Embodiment 1

[0025] Specific embodiments: Example 1 plant gene and Cry2A codon bias analysis:

[0026]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 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 codon usage, 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.

Embodiment 2

[0027] Table 1 Codon usage frequency statistics of various genes Codon amino acid plant gene plant ribosomal protein gene original Cry2ATAA $ 292 5 1TGA $ 465 5 0TAG $ 227 10 0GCT A 7061 80 9GCC A 10110 181 7GCA A 5519 38 9GCG A 7736 89 5TGT C 1663 15 3TGC C 4454 54 1GAT D 8003 65 24GAC D 10376 111 0GAA E 6316 41 15GAG E 13956 200 4TTT F 4189TTC F 8711 108 5GGT G 5567GGC G 5193 49 16GGG G 5470 49 7CAT H 3123 21 8CAC H 4565 72 3ATT I 5047 46 16ATC I 8608 147 5ATA I 27881 6 18AAA K 4817 38 8AAG K 13807 375 1TTA L 1698TTG L 4270 35 9CTT L 53 12CTC L 8852 160 4CTA L 7342 71 3ATG M 8337 88 11AAT N 4672 25 55AAC N 8376 81 13CCT P 4137 43 12CCC P 3893 78 1CCA P 4245 32 11CCG P 4866 62 2CAA Q 4091 31 20CAG Q 8188 109 8CGT R 2145 40 7CGC R 4596 127 0cga R 1239 4CGG R 2808 1AGA R 2676 16 18AGG R 5024 93 63TCT S 3586 26 15TCC S 5603 4TCA S 3536 3296 34 4AGT S 4ACT T 3639 31 21ACC T 6012 122 6ACA T 3558 17 23ACG T 3356 29 7GTT V 5612 61 15GTC V 7167 125 2GTA V 1989 14GTG V 8343 119 6TG...

Embodiment 3

[0028] 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. Example 3: Analysis of the characteristics of the new Cry2A coding sequence synthesized

[0029] 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 i...

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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. Am...

Claims

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Application Information

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