Bacillus thuringiensis cry8 I genes and protein effective for Coleopteran pests, and uses thereof

A technology of Bacillus chrysogenum and Coleoptera, applied in the field of recombinant strains containing cry8I gene, can solve the problems of narrow insecticidal spectrum, single gene variety, and low toxicity

Inactive Publication Date: 2009-04-22
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] Aiming at the shortcomings of the Bt preparation products used in the world to control coleopteran pests and the single gene variety, the pests are easy to develop resistance, the insecticidal spectrum is narrow and the toxicity is low, the present invention isolates and clones new Bt strains from domestic Bt strains. Gene cry8I, the gene expression product has high toxicity to coleopteran pests, and the amino acid sequence of the gene expression product provided by the present invention is significantly different from the known Bt Cry protein

Method used

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  • Bacillus thuringiensis cry8 I genes and protein effective for Coleopteran pests, and uses thereof
  • Bacillus thuringiensis cry8 I genes and protein effective for Coleopteran pests, and uses thereof
  • Bacillus thuringiensis cry8 I genes and protein effective for Coleopteran pests, and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1, identification of cry gene in wild strain BT-SU4

[0065] Two pairs of universal primers were designed according to the conserved regions of cry8 genes. According to the current database analysis, all cry8 genes can be amplified with two pairs of primers, S8Ca5 / S8Ca3 and S8Ca5 / S8Ea3. The primer sequences are as follows:

[0066] S8Ca5: 5'-GATGAGTCCGAATAATCAGAATGAAT-3'

[0067] S8Ca3: 5'-TTACTCTTTCTTCTAACACGAGTTC-3'

[0068] S8Ea3: 5'-TTACTCTACGTCAACAATCAATTC-3'

[0069] Table 2 shows the homologous sequences of these genes and primers, and Table 3 shows the size of the digested fragments of cry8 gene amplified products predicted by this pair of primers. These genes can be identified respectively by this PCR-RFLP method.

[0070]Table 2 The pairing situation of the primers and the conserved regions of cry8 genes and the position of the paired regions on the gene

[0071]

[0072]

[0073] Table 3 PCR amplification products and restriction enzyme lengt...

Embodiment 2

[0079] Cloning of cry8I gene in embodiment 2, wild strain BT-SU4

[0080] A pair of full-length gene primers cry8I5 / cry8I3 was designed to amplify the full-length gene. And the introduction of an EcoRI restriction site at the 5-end for cloning and expression, the sequence of the primer pair cry8I5 / cry8I3 is as follows:

[0081] cry8I5: gg aat tcg atg agt ccg aat aat cag aat

[0082] cry8I3: cgc gtc gac tta cat ttc ttc tac aat caa ttc

[0083] Using the total DNA of the wild strain BT-SU4 as a template, use KOD-PLUS DNA polymerase (product of TOYOBO, Japan, purchased from Beijing Dingguo Biotechnology Co., Ltd.) to perform PCR amplification with the following system:

[0084] 10×PCR buffer 5μL dNTP (10mM) 1μL Primer pair (10mM) 1μL / piece template 1uL KOD-PLUS DNA poly

Synthase (5U / μL) 0.5μL

[0085] Make up to 50 μL with ultrapure water, mix well and centrifuge, add 30 μL of paraffin oil.

[0086] Amplification cycle: denatur...

Embodiment 3

[0110] Example 3, construction of cry8Ia1 full-length gene shuttle vector

[0111] Design primers cry8I5 / cry8I3, in which the 5-primer is added with an EcoR I restriction site, and the 3-end is amplified by high-fidelity KOD-PLUS polymerase. The PCR amplification conditions are 94°C for 1 minute, 54°C 1 minute, 3 minutes at 68°C, 30 cycles, extension at 68°C for 10 minutes, amplified with high-fidelity KOD-PLUS polymerase. The 3.6kbcry8Ia1 full-length fragment was obtained, as attached image 3 Shown in lane 1. After the complete reaction of EcoR I enzyme digestion, electrophoresis was carried out, and the fragment was recovered from the gel, and the Bt-E.Coli shuttle vector pSTK vector (this plasmid came from China Agricultural Biotechnology Laboratory, Institute of Plant Protection, Academy of Sciences, available to the public) Ligation reaction, 4°C, 12 hours; take 5 μL of the ligation product to transform into E.coli recipient strain JM110, and use a kanamycin resistance...

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Abstract

The invention relates to a thuringiensis cry8I gene, protein and application thereof, which belong to the field of biotechnology, wherein the thuringiensis cry8I gene and the protein have high efficiency on coleopteran pests. The thuringiensis cry8I gene which is efficient on the coleopteran pests is obtained through the separation from a wild BT-SU4 strain, a sequence of the thuringiensis cry8I gene is shown in SEQ ID NO1, and an amino acid sequence of a coding of the thuringiensis cry8I gene is shown in SEQ ID NO2. A gene expression product has higher virulence to the coleopteran pests, and can overcome the disadvantages that Bt preparation products and gene species which are used for preventing the coleopteran pests in the world at present are single, make the pests easy to generate the resistance, have narrower insecticidal spectrum and lower virulence and so on; and the new gene can be applied to transforming microorganisms and plants, make the microorganisms and the plants show high virulence to related pests, and overcome and delay the occurrence of the drug resistance of the pests to engineering strains and transgenic plants.

Description

technical field [0001] The invention belongs to the technical field of biological control, and relates to the nucleotide sequence of the cry8I gene highly toxic to coleopteran pests, to the amino acid sequence of a protein highly toxic to coleopteran pests, to a recombinant strain containing the cry8I gene, and to the use of the cry8I gene Gene construction expression vector. Background technique [0002] Scarab belongs to Coleoptera Scarabaeidae (Scarabaeidae), and its larvae (commonly known as grubs, also referred to as "grubs" for short in the present invention hereinafter) are a class of important worldwide distribution underground pests, which can harm food, cotton, oil crops, vegetables, sugar Forage crops, tobacco, grass, flowers, lawn grass, fruit trees and other plants. A large number of surveys have shown that grubs are the most harmful underground pests, among which mainly the larvae of the family Beetleidae and Beetleidae, accounting for more than 70-80% of the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/32C12N15/63C12N1/21C07K14/325A01H1/00A01N63/02A01P7/04C12R1/07
Inventor 闫贵欣束长龙张杰宋福平黄大昉梁影屏
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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