Artificial reconstruction synthetic pesticidal gene as well as encoding protein and use thereof

A technology of insecticidal genes and proteins, applied in the fields of application, genetic engineering, plant genetic improvement, etc., can solve problems such as ecological imbalance and increased resistance of pests

Active Publication Date: 2008-09-03
BIOCENTURY TRANSGENE CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In order to prevent the occurrence of plant pests, it is necessary to spray a large amount of pesticides. The long-term use of chemical pesticides in large quantities will lead to increased resistance of pests, and will kill a large number of their natural enemies, resulting in ecological imbalance and entering a vicious circle.

Method used

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  • Artificial reconstruction synthetic pesticidal gene as well as encoding protein and use thereof
  • Artificial reconstruction synthetic pesticidal gene as well as encoding protein and use thereof
  • Artificial reconstruction synthetic pesticidal gene as well as encoding protein and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1 Synthesis of CmCry2Aa insecticidal gene

[0053] 1. According to the amino acid sequence of Cry2Aa insecticidal protein and cotton codon usage, three gene fragments for constructing CmCry2Aa insecticidal gene were designed and synthesized respectively. Gene synthesis was entrusted to Beijing Aoke Company.

[0054] According to the design, the codon usage of the synthetic CmCry2Aa insecticidal gene is as follows:

[0055]

[0056] Note: The three numbers after the symbol of the amino acid encoded by each codon in the above table represent: number of codons / occurrence frequency in protein (‰) / codon usage (%)

[0057] Domain I (Domain I) DI gene fragment: BamHI-ATG-DI-ApaI, its specific sequence is shown in: SEQNO ID: 5.

[0058] Domain II (DomainII) DII gene fragment: ApaI-DII-SspI, its specific sequence is shown in: SEQ NO ID: 6.

[0059] Domain III (DomainIII) ​​DIII gene fragment: XbaI-HpaI-DIII-SacI, its specific sequence is shown in: SEQ NO ID: 7.

...

Embodiment 2

[0063] Example 2 Construction of CmCry2Aa insecticidal gene plant expression vector M1-BAR-35S-CmCry2Aa

[0064] Have the plasmid CmCry2Aa-pUC57 of complete CmCry2Aa insecticidal gene, further follow as Image 6 The procedure described constructs the plant expression vector M1-BAR-35S-CmCry2Aa. The PCR identification results and enzyme digestion identification results of the screening and identification during the construction of the plant expression vector M1-BAR-35S-CmCry2Aa are as follows: Figure 7 and Figure 8 as shown, Figure 7 Lanes 1, 2 and 3 represent target bands amplified using clones 1, 2 and 3 as templates, and "+" represents target bands amplified using plasmid M1-BAR-35S-CmCry2Aa as a template (positive control), "-" represents that the target band was not amplified using water as a template (negative control); Figure 8 Middle 1 lane is the restriction endonuclease Xba I+SacI cut plasmid M1-BAR-Tnos-Gus, cut off GUS, about 1.8Kbp, 2, 3, 4, 5 and 6 lanes a...

Embodiment 3

[0065] Example 3 Obtaining tobacco with CmCry2Aa insecticidal gene

[0066] Using Agrobacterium-mediated leaf disk method to transform tobacco (a method well known to researchers in the field), and screened by PPT rooting, 46 resistant plants were obtained, the total DNA of resistant tobacco leaves was extracted, and PCR identification was carried out, and 27 positive plants were screened out , and carried out anti-cotton bollworm feeding insect test, each leaf received 5 newly hatched larvae, and after three days the results were as follows: Figure 9 As shown, 1 is the non-transformed CFM CmCry2Aa insecticidal gene tobacco leaves, 2, 3 and 4 are respectively transformed CmCry2Aa insecticidal gene tobacco leaves, the results show that the synthesized CmCry2Aa insecticidal gene has a very prominent anti-cotton bollworm effect. After investigation, the average corrected mortality rate of cotton bollworm was 93.3%.

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Abstract

The invention relates to the field of genetic engineering for plant, which provides an insect killing gene, encoded protein thereof and application of the same. The invention synthesizes CmCry2Aa insect killing protein gene by optimized codon design of cotton, and further obtains Cm1Cry2Aa insect killing protein gene which loses 29 amino acids at N terminal of the encoded protein, and constructs plant expression carrier respectively. Expert evidence of genetic modified tobacco indicates that CmCry2Aa and Cm1Cry2Aa insects killing gene has evident resistance to boll worm.

Description

technical field [0001] The invention relates to the field of plant genetic engineering, in particular to an artificially modified and synthesized insecticidal gene, a plant expression vector constructed by it and its application in the development of insect-resistant transgenic plants. Background technique [0002] Plants are vulnerable to insect pests during their growth, and the annual loss of crops due to insect pests is very large. In order to prevent the occurrence of plant pests, it is necessary to spray a large amount of pesticides. The long-term use of chemical pesticides in large quantities will lead to increased resistance of pests, and will kill their natural enemies in large quantities, leading to ecological imbalance and entering a vicious circle. The use of genetic engineering means to make plants obtain insect resistance has been widely used at present. The genes used for plant insect resistance genetic engineering mainly include: (1) Bacillus thuringiensis (B...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/32C12N15/82C12N5/10C07K14/325A01H1/00A01H5/00
CPCC07K14/325C12N15/8286Y02A40/146
Inventor 崔洪志陈文华杨年松王君丹江辉松
Owner BIOCENTURY TRANSGENE CHINA
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