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Frontiers of Agriculture in China

ISSN 1673-7334

ISSN 1673-744X(Online)

CN 11-5729/S

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Front Agric Chin    2011, Vol. 5 Issue (4) : 423-429    https://doi.org/10.1007/s11703-011-1117-z
RESEARCH ARTICLE
Identification of a novel enhancin-like gene from Bacillus thuringiensis
Dan ZHAO1, Wei GUO1,2,3(), Weiming SUN2, Daqing XU1, Daqun LIU2
1. College of Life Sciences, Agricultural University of Hebei, Baoding 071001, China; 2. College of Plant Protection, Agricultural University of Hebei, Biological Control Center of Plant Diseases and Plant Pests of Hebei Province, Baoding 071001, China; 3. National Engineering Research Center for Agriculture in Northern Mountainous Areas, Baoding 071001, China
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Abstract

An enhancin-like gene was cloned from Bacillus thuringiensis (Bt) strain GS8 isolated from soil samples in china. The sequence analysis revealed that an open reading frame (ORF) of 2202 nucleotides encoding a protein containing 733 amino acids with a molecular mass of 84 kDa. The enhancin-like protein showed 100% identity to Bel protein (FJ644935) and 23%–41% identity to viral enhancin proteins; in the 252 to 261 amino-acid sequence of enhancin-like protein, a conserved metal binding motif (HEIAH) similar to that in the reported bacterial enhancin-like proteins was found (HEXXH in viral enhancin protein), which indicated that the enhancin-like protein belongs to metalloprotease. The purified enhancin-like protein was fed together with Cry9Ea to Spodopera exigua and Trichoplusia ni larvae, but no significant increase in toxicity was observed.
Keywords Bacillus thuringiensis      enhancin-like gene      Cry9Ea protein      synergism     
Corresponding Author(s): GUO Wei,Email:guowei@hebau.edu.cn   
Issue Date: 05 December 2011
 Cite this article:   
Dan ZHAO,Wei GUO,Weiming SUN, et al. Identification of a novel enhancin-like gene from Bacillus thuringiensis[J]. Front Agric Chin, 2011, 5(4): 423-429.
 URL:  
https://academic.hep.com.cn/fag/EN/10.1007/s11703-011-1117-z
https://academic.hep.com.cn/fag/EN/Y2011/V5/I4/423
Strains and plasmidsCharacteristicsOrigin
Strains
E. coli TG1supE hsd⊿5 thi ⊿(lAc-proAB) F’ [traD36 proAB+ lAcIqlAcZ⊿M15]stored in this laboratory
SCS110Rpsl thr leu endA dcm supE44 proABby Dr. Zhang Jie
E. coli BL21F- ompT hsdSB(rB- mB-) gal dcm λ(DE3),stored in this laboratory
HD73-Bt acrystalliferous mutant strainby Dr. Zhang Jie
GS8Wild strainstored in this laboratory
HD9EABt strain, HD73-with pSXY-9EAstored in this laboratory
Plasmids
pET30aKnaRE. coli expression vector, 5.4 kbstored in this laboratory
pSXY422bAmpR ErRBt-E. coli shuttle expression vectorby Dr. Zhang Jie
Tab.1  Strains and plasmids used in this study
Fig.1  PCR amplification of gene of strain GS8. M: marker; 1: PCR product of gene.
Fig.2  Amino acid sequence alignment of enhancing. The highly conserved metalloprotease zinc binding domain (HEXXH) in the region of positions 252 to 261 is shown in the box.
Fig.3  SDS-PAGE showing the induced expression of protein in . M is protein marker; 1-6 are the soluble fraction of ; 7 and 8 are the supernatant and pellet the ultrasonic broken cells, respectively, and 9 is the purification of the protein.
Fig.4  Analysis of enhancin-like protein from HD73-(pSXY422b-) by western blotting. M is protein marker; 1 is HD73-(pSXY422b-), and 2 is HD73–(–).
Fig.5  A and B are the synergistic effects between the enhancin-like protein and Cry9Ea toward to and .
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[1] Tinghui LIU, Wei GUO, Weiming SUN, Yongxiang SUN. Biological characteristics of Bacillus thuringiensis strain Bt11 and identification of its cry-type genes[J]. Front Agric Chin, 2009, 3(2): 159-163.
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