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Bt Research (Online) 2010, Vol.1 No.2
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- 5 -
was lacking in the strain
Bt
w015
-
1. Although the
PCR amplicon of W015
-
1 is the same size as that
of HD73, the RFLP patterns of the strains are
different in size and number. The results indicated
that
Bt
W015
-
1 has a different cry genotype than
that of standard strain HD73. Purified PCR
amplicons were ligated into pMD18
-
T vector to
construct the recombinant plasmid pMDK2 and
pMDK3 for sequencing (Beijing Genome Institute,
Beijing, China). The sequence analysis revealed that
deduced amino acid residues of the amplicons
generated by the above primer was highly similar to
cry1Ac1
, which implied that the W015
-
1 strain
contains a
cry1Ac
type gene.
1.5 Clonine of
cry1Ac
gene
In order to obtain the full length sequence of Cry1Ac
from W015
-
1, inverted PCR was used to amplify
the sequences through primer pair cryI5/cryI3
designed by primer software based on the sequences
of plasmids pMDK2 and pMDK3.
The restriction endonucleases
Ned
Ⅰ
,
Sal
Ⅰ
,
Bgl
Ⅱ
and
Bam
H
Ⅰ
were used to to completely digest
the plasmids of W015
-
1, and then inactivated the
restriction enzymes were inactivated in a 65
℃
water bath for 15 min, followed by the addition of
T4 DNA ligase to randomly connect the digested
fragments. The inverse PCR produced the 1.6 kb
fragment (Figure 5) based on the
Ned
Ⅰ
digestion
fragment as template. This product was ligated into
the vector pMD18
-
T to construct the recombinant
plasmid pMDIS for sequencing. The sequences of
recombinant pMDK2, pMD1K3 and pMDIS were
assembled into a spliced DNA sequence of 3,772 bp,
which contained a 3,534 bp open reading frame
(ORF). The primer pairs of E1A5/E1A3 were designed
based on the assembled sequence to amplify the full
length sequence of the ORF of about 3.5 kb (Figure 6).
This further confirmed that the
cry1Ac
gene existed
in the strain W015
-
1.
The sequence of this gene deposited in the GenBank
with accession number EU282379 was designated
as
cry1Ac22
based on the nomenclature system
proposed by Crickmore et al (1998). The coding
sequence of
cry1Ac22
with 3,534 bp in length
encodes a putatively weak acidic polypeptide of
1,178 amino acid residues with estimated molecular
weight of 133 kD and iso-electric point of 5.04,
which includes 30.90% hydrophilic amino acids,
32.64% hydrophobic amino acids, 13.83% acidic
Figure 5 Full length
cry1Ac
gene amplified by inverted PCR
Note: A: Agarose gel electrophoresis of plasmid DNA from
W015
-
1; 1,2,3,4 are digested fragments of W015
-
1 plasmid
DNA by
Bam
H
Ⅰ
,
Bgl
Ⅱ
,
Sal
Ⅰ
and
Ned
Ⅰ
respectively; B:
Products of Inverted PCR, 1,2,3,4 represent that the DNA
templates come from the W015
-
1 plasmid DNA by
Bam
H
Ⅰ
,
Bgl
Ⅱ
,
Sal
Ⅰ
and
Ne
d
Ⅰ
respectively
Figure 6 Full sequence of
cry1Ac22
gene amplified by PCR
amino acids and 11.43% basic amino acids. The
deduced amino acid sequence of
cry1Ac22
has 99%
similarity to
cry1Ac1
, whereas three sites of 233
(T/R), 448 (M/I) and 1158 (K/E) are differences
existing between
cry1Ac22
and
cry1Ac1
. Multiple
sequence alignment and conserved domain of
cry1Ac22
were generated by using clustalW and
proDom programs (http://www.ebi.ac.uk/Tools/clustal
w2/index.html, http://prodom.prabi.fr/prodom/ current/
html/form.php). The results indicated that the
cry1Ac22
toxic protein has three protein domains
and five conserved blocks that are typical features
of cry 1A proteins (Figure 7). Three-dimensional
structure prediction revealed that Cry1Ac22, whose