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Research Article Open Access
Characterization of a New Highly Toxic Isolate of
Bacillus thuringiensis
from the
Diapausing Larvae of Silkworm and Identification of
cry1A 22
Gene
Liu Xie
1,2*
, Wenfei Zhang
1,2*
, Zhuoming Liu
1,2
, Yugeng Cai
4
, Youzhi Li
1
, Xuanjun Fang
1,2,3
1 College of Life and Technology Science, Guangxi University, Nanning, 530004, P.R.China
2 Hainan Provincial Key Lab for Crop Molecular Breeding, Sanya, 572025, P.R.China
3 Hainan Institute of Tropical Agricultural Resources, Sanya, 572025, P.R.China
4 Jiaxing Silkworm Seed Station, Jiaxing, 314000, P.R.China
* These authors contributed equally
Corresponding author, xuanjunfang@hitar.org;
Authors
Bt Research 2010, Vol 1 No 2
DOI: 10.5376/bt.2010.01.0002
Received: 15 Aug., 2010
Accepted: 19 Sep., 2010
Published: 28 Dec., 2010
This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
Preferred citation for this article:
Xie et al., 2010, Characterization of a New Highly Toxic Isolate of
Bacillus thuringiensis
from the Diapausing Larvae of Silkworm and Identification of
cry1A 22
Gene, Bt Research (online), Vol.1 No.2 (DOI: 10.5376/bt.2010.01.0002)
Abstract
We have isolated 218
Bacillus
isolates from thedissected guts of 100 diapausing larvae of the silkworm,
Bombyx mori
,
collected from silkworm farmers in the Hangjiahu area of Zhejiang Province. Six isolates were identified as
Bacillus thuringiensis
strains. The strain named as W015
-
1 is highly toxic to the lepidopteran
Plutella xylostella
, and is deposited in the HITAR
Bacillus
Collections with Accession No. 20050509W015. Strain W015
-
1 can synthesize bipyramidal crystals during sporulation as observed
under light and scanning electron microscope. SDS-PAGE analysis showed that the dominant protein has a molecular mass of about
130 kD. The plasmid profile was revealed based on the technology of pulsed field gel electrophoresis (PFGE). W015
-
1 has a large
plasmid profile very similar in size, number and banding pattern to the reference strain
Btk
HD73, and is completely different to
Btk
HD1 and
Bti
AND508. When we employed RFLP-PCR approach to identify the genotype of the strains, the results indicated that
Bt
strain W015
-
1 has a cry1A genotype with different enzyme cutting sites compared to the reference strain HD73. The full coding
sequence of the crystal toxin was cloned (GenBank accession number EU282379) by combining the techniques of PCR-RFLP and
inverted PCR and was designated as
cry1Aa22
according to the nomenclature system proposed by Crickmore et al. Sequence analysis
revealed that this gene contained an open reading frame of 3,534 nucleotides encoding a protein of 1,178 amino acid residues
containing three typical toxin domains, and is highly homologous with the Cry1Ac family. There are three existing differences with
the sequence of known cry 1Ac1, at 233 (T/R), 448 (M/I) and 1158 (K/E). We ligated the
cry1Ac22
into
E. coli
expression vector
pQE30 to construct pQE30
-
cry1Ac22
and then the recombinant plasmids were transformed into
E. coli
M15 to express an inclusion
protein of about 133 kD. The inclusion of Cry1Ac22 can be hydrolysed to a trypsin-activated form with a molecular weight of about
80 kD. Larvicidal assays of the trypsin-activated form of Cry1Ac22 were carried out and was demonstrated high insecticidal activity
against larvae of
Plutella xylostella
(LC
50
: 4.135×10
8
cfu/mL; 95% FL: 3.368~5.122×10
8
cfu/mL), which was much higher than that
of model strain HD 73. W015
-
1 and the reference strains are dissimilar with differing in plasmid profiles, cry genotypes and crystal
proteins. Thus, it is believed that
Bt
W015
-
1 could be a potential biopesticide alternative to
Btk
.
Keywords
Silkworm;
Bombyx mori
;
B. thuringensis
; Parasporal crystal toxin; Cry genotype;
cry1Ac22
; Plasmid profile;
Insecticidal activity
Background
With more than 160,000 species, Lepidoptera is
recognized as the second most bio-diverse group of
insects after Coleoptera, it includes many of the